Methods and compositions for treating autoimmune and inflammatory conditions

ABSTRACT

Described herein are compositions and methods for inhibiting an inflammatory or autoimmune response and for inducing immune tolerance in a subject in need thereof comprising administering to the subject a therapeutically effective amount of an antigen presenting cell (APC)-targeted antibody operatively linked to IL-10 or a fragment thereof. The compositions and methods described herein are useful for treating inflammatory and autoimmune disorders.

This application is a national phase application under 35 U.S.C. § 371of International Application No. PCT/US2015/031117, filed May 15, 2015,which claims the benefit of priority to U.S. Provisional PatentApplication Ser. No. 61/994,239, filed May 16, 2014, and U.S.Provisional Patent Application Ser. No. 62/014,504, filed Jun. 19, 2014,the entire contents of each of which are hereby incorporated byreference in their entirety.

The invention was made with government support under Grant No.1R56AI105066-01 and Grant No. 2. 1R01AI105066-01A1 awarded by theNational Institute of Allergy and Infectious Disease/National Institutesof Health. The government has certain rights in the invention.

BACKGROUND OF THE INVENTION 1. Field of the Invention

The present invention relates generally to the field of medicine. Moreparticularly, it concerns pharmaceutical compositions for enhancingtolerance to antigens and for treating inflammatory and autoimmunedisorders.

2. Background

Autoimmune and inflammatory diseases arise from an abnormal immuneresponse of the body against substances and tissues normally present inthe body. This may be restricted to certain organs (e.g., in autoimmunethyroiditis) or involve a particular tissue in different places (e.g.,Goodpasture's disease which may affect the basement membrane in both thelung and the kidney). Autoimmune and auto-inflammatory diseases affectup to 50 million people in America alone, and the cause of autoimmunityremains unknown.

The treatment of these diseases is typically withimmunosuppression-medication that decreases the immune response.Conventional immunotherapies using immunosuppressants, such ascyclosporine, tacroliums, methotrexate or anti-TNFa/IL-6non-specifically suppress the function of T cell includingnon-pathogenic T cells in the host. Therefore, treatment with theseimmunesuppressants often results in the development of severe infectionsand sometimes leads to the lethal consequences. There is a need in theart for therapeutics that treat autoimmune responses without globalimmunosuppression.

SUMMARY OF THE INVENTION

This disclosure fulfills a need in the art by providing methods andcompositions for delivering the anti-inflammatory cytokine, IL-10, toantigen presenting cells (APCs) to suppress and alter thepathophysiologic functions of APCs in the subjects using APC-targetedantibody operatively linked to IL-10 or a fragment thereof. Targeteddelivery of anti-inflammatory cytokines to the APCs in the patients isexpected to result in more effective and pro-longed immune tolerance inthe patients. Accordingly, aspects of the disclosure relate to a methodfor inhibiting an inflammatory or autoimmune response in a subject inneed thereof comprising administering to the subject a therapeuticallyeffective amount of an antigen presenting cell (APC)-targeted antibodyoperatively linked to IL-10 or a fragment thereof.

In some embodiments, the disclosure relates to a method for preventingor treating graft versus host disease in a subject in need thereofcomprising administering to the subject a therapeutically effectiveamount of DC-ASGPR operatively linked to IL-10 or a fragment thereof.

Further aspects relate to a method of inducing immune tolerance in asubject in need thereof comprising administering to the subject atherapeutically effective amount of an APC-targeted antibody operativelylinked to IL-10 or a fragment thereof. Other aspects relate to a methodof suppressing a T cell response in a subject in a subject having or atrisk of developing an inflammatory response by administering to thesubject a therapeutically effective amount of an APC-targeted antibodyoperatively linked to IL-10 or a fragment thereof.

The term “operatively linked” refers to a situation where two componentsare combined to form the active complex prior to binding at the targetsite. For example, an antibody conjugated to one-half of acohesin-dockerin complex and a cytokine (e.g. IL-10) or other molecule(e.g. antigen) complexed to the other one-half of the cohesin-dockerincomplex are operatively linked through complexation of the cohesin anddockerin molecules. The term operatively linked is also intended torefer to covalent or chemical linkages that conjugate two moleculestogether.

Yet further aspects relate to methods and compositions for treatingundesired and/or abnormal immune responses without non-specificsuppression of the host immune system. In particular, an anti-DC-ASGPRantibody or antigen binding fragment thereof can be used in compositionsand methods described herein for generating anti-pathogenicantigen-specific T regulatory cells and/or for decreasing pathogenic Tcell responses.

The term “anti-pathogenic antigen-specific T regulatory cells” refers toT cells with beneficial and therapeutic properties. In one embodiment,the anti-pathogenic antigen-specific T regulatory cells arealloantigen-specific T regulatory cells. In another embodiment, theanti-pathogenic antigen-specific T regulatory cells is one that producesIL-10. The anti-pathogenic antigen-specific T regulatory cells may alsobe a CD4+ T cell.

The term pathogenic T cell responses refers to abnormal or undesired Tcell responses that contribute to the pathology of autoimmune disease orto the pathology of graft versus host disease (GVHD) or graft rejection.In one embodiment, the pathogenic T cell response is an allogeneic Tcell response. In a further embodiment, the pathogenic T cell responsecomprises allogeneic CD4+ and CD8+ T cells. In one embodiment, thepathogenic T cell response is one that comprises immune cells of thetissue graft.

A further aspect of the disclosure relates to a method for preventing ortreating GVHD in a subject in need thereof comprising administering tothe subject an anti-DC-ASGPR antibody or antigen binding fragmentthereof.

Graft-versus-host disease (GVHD) is a common complication following anallogeneic tissue transplant. It is commonly associated with stem cellor bone marrow transplant but the term also applies to other forms oftissue graft. Immune cells (white blood cells) in the tissue (the graft)recognize the recipient (the host) as “foreign”. The transplanted immunecells then attack the host's body cells. GVHD may also occur after ablood transfusion if the blood products used have not been irradiated.

In another instance, the disclosure describes a method for preventing ortreating graft rejection in a subject in need thereof comprisingadministering to the subject an anti-DC-ASGPR antibody or antigenbinding fragment thereof.

Graft rejection occurs when transplanted tissue is rejected by therecipient's immune system, which destroys the transplanted tissue. Graftrejection may also be referred to as transplant rejection or host versusgraft disease.

In certain embodiments, the antibody or antigen binding fragmentspecifically binds to DC-ASGPR and activates DC-ASGPR.DC-asialoglycoprotein receptor (DC-ASGPR) is a scavenger receptorcarrying an immunoreceptor tyrosine-based activation motiflike motif.ASGPR may also be known as ASGR1, ASGPR1, CLEC4H1, and HL-1. In oneembodiment, the antibody or antigen binding fragment thereof binds tohuman DC-ASGPR.

In some embodiments, the APC-targeted antibody targets one or more APCsof the group Langerhans cells, macrophages, dendritic cells, B cells,and peripheral blood mononuclear cells. In further embodiments, theAPC-targeted antibody is selected from an antibody that specificallybinds to MHC class I, MHC class II, CD1d, CD2, CD3, CD4, CD8, CD11b,CD14, CD15, CD16, CD19, CD20, CD29, CD31, CD40, CD43, CD44, CD45, CD54,CD56, CD57, CD58, CD83, CD86, CMRF-44, CMRF-56, DCIR, DC-ASGPR, CLEC-6,CD40, BDCA-2, MARCO, DEC-205, mannose receptor, Langerin, DECTIN-1,B7-1, B7-2, IFN-γ receptor, IL-2 receptor, ICAM-1, Fc γ receptor, LOX-1,and ASPGR.

In other embodiments, the APC-targeted antibody targets Langerhanscells. One example of an APC-targeted antibody to Langerhans cells isanti-Langerin. In further embodiments, the APC-targeted antibody targetsmacrophages. For example, the APC-targeted antibody may be anti-MARCO.

In yet further embodiments, the APC-targeted antibody targets one ormore APCs of the group dendritic cells, B cells, and macrophages. Inspecific embodiments, the APC-targeted antibody targets dendritic cells.In some embodiments, the APC-targeted antibody comprises anti-CD40. Infurther embodiments, the anti-CD40 antibody comprises anti-CD40 clone12E12 or fragments thereof. As shown in Example 1, anti-CD40(12E12)-IL-10 suppressed the expression of CD86. In some embodiments,the anti-CD40 antibody comprises one or more CDRs having a sequence ofSEQ ID NOS:31-33 and 37-39. In other embodiments, the anti-CD40 antibodycomprises a heavy chain comprising one or more CDRs of SEQ ID NOS:31-33.In further embodiments, the anti-CD40 antibody comprises a light chaincomprising one or more CDRs of SEQ ID NOS:37-39.

In specific embodiments, the anti-CD40 antibody is a humanized antibodycomprising a heavy chain comprising three CDRs, wherein CDR1 comprisesSEQ ID NO:31, CDR2 comprises SEQ ID NO:32, and CDR3 comprises SEQ IDNO:33. In further embodiments, the anti-CD40 antibody is a humanizedantibody comprising a light chain comprising three CDRs, wherein CDR1comprises SEQ ID NO:37, CDR2 comprises SEQ ID NO:38, and CDR3 comprisesSEQ ID NO:39.

In some embodiments, the APC-targeted antibody comprises anti-DC-ASGPRor anti-Dectin-1. The anti-DC-ASGRP or anti-Dectin-1 may be one known inthe art or described herein. In some embodiments the antibody comprisesa variable region comprising an amino acid sequence selected from thesequences of SEQ ID NOs: 3, 8, 62, 64, 66, or 68. In some embodiments,the antibody comprises a heavy or light chain with an amino acidsequence selected from the sequences of SEQ ID NOs: 1, 7, 61, 63, 65,67, or 69-72. In some embodiments, the antibody comprises one or moreCDRs from the variable region, heavy chain, or light chain of SEQ IDNOs: 1, 3, 7, 8, 61, 62, 63, 64, 65, 66, 67, or 68-72.

In some embodiments, the APC-targeted antibody comprises anti-DCIR. Inspecific embodiments, anti-DCIR antibody comprises anti-DCIR clone 9E8or fragments thereof. In further embodiments, the anti-DCIR antibodycomprises one or more CDRs having a sequence of SEQ ID NOS:18-20 or24-26. In other embodiments, the anti-DCIR antibody comprises a heavychain comprising one or more CDRs of SEQ ID NOS:18-20. In yet furtherembodiments, the anti-CD40 antibody comprises a light chain comprisingone or more CDRs of SEQ ID NOS:24-26.

Certain aspects of the disclosure relate to a method for inhibiting aninflammatory or autoimmune response or for inducing tolerance in asubject in need thereof comprising administering to the subject atherapeutically effective amount of an anti-CD40 antibody operativelylinked to IL-10 or a fragment thereof, wherein the anti-CD40 is ahumanized antibody having three heavy chain CDRs comprising an aminoacid sequence of SEQ ID NO:31 (CDR1), SEQ ID NO:32 (CDR2), and SEQ IDNO:33 (CDR3) and three light chain CDRs comprising an amino acidsequence of SEQ ID NO:37 (CDR1), SEQ ID NO:38 (CDR2), and SEQ ID NO:39(CDR3).

Further aspects relate to a method for inhibiting an inflammatory orautoimmune response or for inducing tolerance in a subject in needthereof comprising administering to the subject a therapeuticallyeffective amount of an anti-DCIR antibody operatively linked to IL-10 ora fragment thereof, wherein the anti-DCIR is a humanized antibody havingthree heavy chain CDRs from the variable region of anti-DCIR 9E8 heavychain (SEQ ID NO:17) and three light chain CDRs from the variable regionof anti-DCIR 9E8 light chain (SEQ ID NO:23).

Further aspects relate to a method for inhibiting an inflammatory orautoimmune response in a subject in need thereof comprisingadministering to the subject a therapeutically effective amount of ananti-DC-ASGPR antibody operatively linked to IL-10 or a fragmentthereof, wherein the anti-DC-ASGPR antibody is a humanized antibodyhaving three heavy chain CDRs and three light chain CDRs from thevariable regions of an anti-DC-ASGPR heavy chain and light chain pairselected from SEQ ID NO:3 and 8; SEQ ID NO:58 and 60; SEQ ID NO:62 and64; or SEQ ID NO:66 and 68; or is a humanized antibody having threeheavy chain CDRs and three light chain CDRs from the heavy and lightchains of an anti-DC-ASGPR heavy chain and light chain pair selectedfrom SEQ ID NO:69 and 70 and SEQ ID NO:71 and 72.

In some embodiments, the APC-targeted antibody or antibody conjugate orantigen binding fragment thereof comprises an amino acid sequence thatis at least or at most 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or99% identical or similar (or any derivable range therein) to anAPC-targeted antibody or antigen binding fragment of any of SEQ IDNOS:1, 2, 3, 7, 8, 10, 11, 13, 15-20, 22-26, 28-33, 35-39, or 45-114 (orany range derivable therein). In further embodiments, the APC-targetedantibody conjugate or antigen binding fragment thereof comprises avariable region comprising an amino acid sequence that is at least or atmost 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% (or any rangederivable therein) identical or similar to the APC-targeted antibodyvariable region described herein as SEQ ID NOS: 3, 8, 17, 23, 30, 36,46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 79, 81, 83, 85, 87, 89,108, 110, 112, and 114. In further embodiments, the antibody comprises aCDR having an amino acid sequence corresponding to a CDR in any one ofSEQ ID NOS: 2, 3, 7, 8, 11, 13, 16, 17-20, 22-26, 29-33, 35-39, or45-114 (or any derivable range therein). In some embodiments, theantibody comprises the CDRs of SEQ ID NOS:18-20, 24-26, 31-33, or 37-39.In further embodiments, the APC-targeted antibody or antigen bindingfragment thereof comprises a heavy or light chain amino acid sequencethat is at least or at most 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%,or 99% (or any range derivable therein) identical or similar to theAPC-targeted antibody or antigen binding fragment of any of SEQ IDNOs:1, 2, 7, 10, 11, 13, 15, 16, 22, 28, 29, 35, 45, 47, 49, 51, 53, 55,57, 59, 61, 63, 65, 67, 69-78, 80, 82, 84, 86, 88, 90-107, 109, 111, or113. In certain embodiments, the antibody conjugate or antigen bindingfragment thereof comprises CDR1, CDR2, and/or CDR3 from the heavy and/orlight chain variable region of a APC-targeted antibody described herein.In certain embodiments, the antibody conjugate or antigen bindingfragment thereof comprises all three CDRs from the light chain variableregion and/or all three CDRs from the heavy chain variable region of aAPC-targeted antibody described herein.

In certain embodiments, the antibody or antigen binding fragmentspecifically binds to DC-ASGPR and activates DC-ASGPR.DC-asialoglycoprotein receptor (DC-ASGPR) is a scavenger receptorcarrying an immunoreceptor tyrosine-based activation motiflike motif.ASGPR may also me known as ASGR1, ASGPR1, CLEC4H1, and HL-1. In oneembodiment, the antibody or antigen binding fragment thereof binds tohuman DC-ASGPR.

In some embodiments, the antibody or antigen binding fragment of themethods and compositions described herein is an anti-DC-ASGPR antibodyand comprises an amino acid sequence that is at least or at most 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% (or any derivable rangetherein) identical or similar to the DC-ASGPR antibody or antigenbinding fragment of any of SEQ ID NO: 2, 3, 7, 8, and 61-72 (or anyrange derivable therein). In a further embodiment, the DC-ASGPR antibodyor antigen binding fragment thereof may include a polypeptide, peptide,or protein that is, is at least, or is at most 70%, 75%, 80%, 85%, 90%,95%, 96%, 97%, 98%, or 99% (or any range derivable therein) identical orsimilar to an ASGPR binding polypeptide, such as Anti-ASGPR_49C11_7H(heavy chain), SEQ ID NO:2; Anti-ASGPR_49C11_7K (light chain), SEQ IDNO:7; anti-hASGPR_6.3H9.1D11H (heavy chain), SEQ ID NO:69;anti-hASGPR_6.3H9.1D11K (light chain), SEQ ID NO:70;anti-hASGPR_5H8.1D4H (heavy chain), SEQ ID NO:71; anti-hASGPR_5H8.1D4K(light chain), SEQ ID NO: 72; Anti-ASGPR_4G2.2_ (heavy chain), SEQ IDNO: 57; Anti-ASGPR_4 G2.2_ (light chain), SEQ ID NO: 59;Anti-ASGPR-5F10H (heavy chain), SEQ ID NO:61; Anti-ASGPR-5F10H (lightchain), SEQ ID NO: 63; Anti-ASGPR1H11 (heavy chain), SEQ ID NO: 65; orAnti-ASGPR1H11 (light chain). SEQ ID NO: 67. In further embodiments, theDC-ASGPR antibody or antigen binding fragment thereof comprises avariable region comprising an amino acid sequence that is at least or atmost 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical orsimilar to the DC-ASGPR antibody or antigen binding fragment of any ofSEQ ID NOs: 3, 8, 62, 64, 66, and 68. In some embodiments, the antibodycomprises at least or exactly one, two, or all three CDRs of a variableregion from a heavy or light chain amino acid sequence selected from SEQID NO:2, 7, 57, 59, 61, 63, 65, 67, and 69-72. In some embodiments, theantibody comprises at least or exactly one, two, or all three CDRs of avariable region from a heavy or light chain variable region amino acidsequence selected from SEQ ID NO:3, 8, 58, 60, 62, 64, 66, and 68. Infurther embodiments, the antibody comprises at least or exactly 1, 2, 3,4, 5, or 6 (or any derivable range therein) CDRs from a heavy and lightchain antibody fragment selected from SEQ ID NOS: 2 and 7, SEQ ID NOS:57 and 59; SEQ ID NOS: 61 and 63; SEQ ID NOS: 65 and 67; SEQ ID NOS: 69and 70; or SEQ ID NOS: 71 and 72. In some embodiments, the antibodycomprises at least or exactly 1, 2, 3, 4, 5, or 6 (or any derivablerange therein) CDRs from a heavy and light chain variable regionantibody fragment selected from SEQ ID NOS: 3 and 8, SEQ ID NOS: 58 and60; SEQ ID NOS: 62 and 64; or SEQ ID NOS: 66 and 68.

The ASGPR antibody or antigen binding fragments described herein mayinclude 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54,55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72,73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90,91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more variant amino acidswithin at least, or at most 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69,70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87,88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104,105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118,119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132,133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146,147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160,161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174,175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188,189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202,203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216,217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230,231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244,245, 246, 247, 248, 249, 250, 300, 400, 500, 550, 1000 or morecontiguous amino acids, or any range derivable therein, of SEQ ID NO: 2,3, 7, 8, and 61-72.

The APC-targeted antibody conjugate or antigen binding fragmentsdescribed herein may include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67,68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85,86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or morevariant amino acids (or any range derivable therein) within at least, orat most 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56,57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74,75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92,93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108,109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122,123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136,137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150,151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164,165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178,179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192,193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206,207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220,221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234,235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248,249, 250, 300, 400, 500, 550, 1000 or more contiguous amino acids, orany range derivable therein, of any of SEQ ID NOs:1-5, 7-8, 10-11, 13,15-20, 22-26, 28-33, 35-39, or 45-114.

Embodiments are provided in which the APC-targeted antibody or antigenbinding fragments comprises one or more CDR domains from an antibodythat specifically binds to an antigen presenting cell surface protein.In particular embodiments, the APC-targeted antibody or antigen bindingfragment thereof comprises one, two, three, four, five, six, or more CDRdomains from among the VH or VL domain of the monoclonal antibodieslisted herein in SEQ ID NOS: 3, 8, 17, 23, 30, 36, 46, 48, 50, 52, 54,56, 58, 60, 62, 64, 66, 68, 79, 81, 83, 85, 87, 89, 108, 110, 112, and114. In certain aspects, the APC-targeted antibody or antigen bindingfragment thereof comprises six CDR domains from among the VH or VLdomains of the monoclonal antibodies: anti-Dectin-1 clone 11B6.4,15E2.5, or 2D8.2D4; ASGPR clone 49C11, 4G2.2, 5F10, 1H11, 6.3H9.1D11, or5H8.1D4; anti-CD40 clone 12E12, 12B4.2C10, 24A3, or 11B6.1C3; anti-Lox-1clone 11C8, 10F9, or 15C4; anti-DCIR clone 24A5.4A5, 24E7.3H9, 29E9.2E2,29G10.3D9, 31A6.IF5, 3C2.2D9, 6C8.1G9, 9E8, or 2C9; or anti-Langrinclone 15B10 or 2G3. In some embodiments, the APC-targeted antibody orantigen binding fragment thereof comprises a sequence at least or atmost 70%, 75%, 80%, 85%, 90%, 95%, or 99% (or any range derivabletherein) identical to the VH or VL domain of the monoclonal antibodies:anti-Dectin-1 clone 11B6.4, 15E2.5, or 2D8.2D4; ASGPR clone 49C11,4G2.2, 5F10, 1H11, 6.3H9.1D11, or 5H8.1D4; anti-CD40 clone 12E12,12B4.2C10, 24A3, or 11B6.1C3; anti-Lox-1 clone 11C8, 10F9, or 15C4;anti-DCIR clone 24A5.4A5, 24E7.3H9, 29E9.2E2, 29G10.3D9, 31A6.IF5,3C2.2D9, 6C8.1G9, 9E8, or 2C9; or anti-Langrin clone 15B10 or 2G3.Embodiments are provided in which the APC-targeted antibody or antigenbinding fragment thereof comprises the VH domain from the monoclonalantibodies listed herein and/or the VL domain from the monoclonalantibodies listed herein. In further embodiments, the monoclonalantibody is selected from: anti-Dectin-1 clone 11B6.4, 15E2.5, or2D8.2D4; ASGPR clone 49C11, 4G2.2, 5F10, 1H11, 6.3H9.1D11, or 5H8.1D4;anti-CD40 clone 12E12, 12B4.2C10, 24A3, or 11B6.1C3; anti-Lox-1 clone11C8, 10F9, or 15C4; anti-DCIR clone 24A5.4A5, 24E7.3H9, 29E9.2E2,29G10.3D9, 31A6.IF5, 3C2.2D9, 6C8.1G9, 9E8, or 2C9; or anti-Langrinclone 15B10 or 2G3.

In certain embodiments, the APC-targeted antibody or antigen bindingfragment thereof is recombinant. In certain aspects, the recombinantpolypeptide comprises at least 90%, 95%, or 99% of one or more CDRdomains from the VH or VL domain of the anti-Dectin-1 clone 11B6.4,15E2.5, or 2D8.2D4; ASGPR clone 49C11, 4G2.2, 5F10, 1H11, 6.3H9.1D11, or5H8.1D4; anti-CD40 clone 12E12, 12B4.2C10, 24A3, or 11B6.1C3; anti-Lox-1clone 11C8, 10F9, or 15C4; anti-DCIR clone 24A5.4A5, 24E7.3H9, 29E9.2E2,29G10.3D9, 31A6.IF5, 3C2.2D9, 6C8.1G9, 9E8, or 2C9; or anti-Langrinclone 15B10 or 2G3 monoclonal antibodies. In some embodiments, therecombinant polypeptide comprises two, three, four, five, six, or moreCDR domains from the VH or VL domain of the anti-Dectin-1 clone 11B6.4,15E2.5, or 2D8.2D4; ASGPR clone 49C11, 4G2.2, 5F10, 1H11, 6.3H9.1D11, or5H8.1D4; anti-CD40 clone 12E12, 12B4.2C10, 24A3, or 11B6.1C3; anti-Lox-1clone 11C8, 10F9, or 15C4; anti-DCIR clone 24A5.4A5, 24E7.3H9, 29E9.2E2,29G10.3D9, 31A6.IF5, 3C2.2D9, 6C8.1G9, 9E8, or 2C9; or anti-Langrinclone 15B10 or 2G3 monoclonal antibodies.

In some embodiments, a recombinant polypeptide comprises i) CDR1 (SEQ IDNO:37), CDR2 (SEQ ID NO:38), and/or CDR3 (SEQ ID NO:39) from thevariable light chain of anti-CD40 12E12; and/or ii) CDR1 (SEQ ID NO:31),CDR2 (SEQ ID NO:32), and/or CDR3 (SEQ ID NO:33) from the variable heavychain of 12E12. In some embodiments, a recombinant polypeptide comprisesi) CDR1, CDR2, and/or CDR3 from the variable light chain of anti-DCIR9E8; and/or ii) CDR1, CDR2, and/or CDR3 from the variable heavy chain of9E8.

Certain aspects are directed to methods of inhibiting an inflammatoryresponse or inducing tolerance in a subject in need thereof comprisingadministering to the subject an effective amount of one or moreAPC-targeted antibody or antigen binding fragment thereof operativelylinked to IL-10. The antibody can be a purified polyclonal antibody, apurified monoclonal antibody, a recombinant polypeptide, or a fragmentthereof. In certain aspects the antibody is humanized or human. In stillfurther aspects the antibody is a recombinant antibody segment. Incertain aspects a monoclonal antibody includes one or more ofanti-Dectin-1 clone 11B6.4, 15E2.5, or 2D8.2D4; ASGPR clone 49C11,4G2.2, 5F10, 1H11, 6.3H9.1D11, or 5H8.1D4; anti-CD40 clone 12E12,12B4.2C10, 24A3, or 11B6.1C3; anti-Lox-1 clone 11C8, 10F9, or 15C4;anti-DCIR clone 24A5.4A5, 24E7.3H9, 29E9.2E2, 29G10.3D9, 31A6.IF5,3C2.2D9, 6C8.1G9, 9E8, or 2C9; or anti-Langrin clone 15B10 or 2G3. Anantibody can be administered at a dose of 0.1, 0.5, 1, 5, 10, 50, 100 mgor μg/kg to 5, 10, 50, 100, 500 mg or μg/kg, or any range derivabletherein.

The methods described herein provide a dose sparing effect such that thetargeted delivery of IL-10 requires a smaller amount or dose to achievethe same effect as a non-targeted IL-10. In certain embodiments, thetherapeutically effective amount of the APC-targeted antibodiesoperatively linked to IL-10 is at least 5, 10, 20, 50, 100, 500, or 1000fold less than the dose of non-targeted IL-10. In further embodiments,the therapeutically effective amount of the APC-targeted antibodiesoperatively linked to IL-10 is greater than 50%, greater than 75%,greater than 80%, greater than 90% or greater than 99% less than theeffective amount of the dose of non-targeted IL-10. The therapeuticallyeffective amount of non-targeted IL-10 is known in the art, and may varydepending on the disease to be treated. In certain embodiments, theeffective amount of non-targeted IL-10 is 1, 5, 10, or 20 μg/kg. In oneembodiment, the effective amount of non-targeted IL-10 is 5 μg/kg. Inother embodiments, the therapeutically effective amount of theAPC-targeted antibodies operatively linked to IL-10 is at least 5 foldless than the dose of non-targeted IL-10.

In certain embodiments, the antibody is a human antibody, humanizedantibody, recombinant antibody, bi-specific antibody, chimeric antibody,a nanobody, a DARPin, an antibody derivative, a veneered antibody, adiabody, a monoclonal antibody, or a polyclonal antibody. In a specificembodiment, the antibody is a humanized antibody.

In certain embodiments, the antibody is a non-naturally occurringantibody. In some embodiments, the antibody is non-naturally occurringsince it comprises at least two polypeptide segments from differentsources. The different sources may be different mammals, such as humanand mouse, for example.

In some embodiments of the methods described herein, the subject is ahuman subject. The term “subject,” “individual” or “patient” is usedinterchangeably herein and refers to a vertebrate, for example aprimate, a mammal or preferably a human. Mammals include, but are notlimited to equines, canines, bovines, ovines, murines, rats, simians,humans, farm animals, sport animals and pets.

In some embodiments, the subject is one that has an autoimmune diseaseor an inflammatory disorder. The autoimmune disease or inflammatorydisorder may be one known in the art and/or described herein. In someembodiments, the autoimmune disease or inflammatory disorder is selectedfrom rheumatoid arthritis, allergy, asthma, systemic onset juvenilearthritis, inflammatory bowel disease, systemic lupus erythematosus,multiple sclerosis, type 1 diabetes, graft rejection, graft versus hostdisease, colitis, and Crohn's disease.

In some embodiments, the subject is at risk for the development of adisease mediated by a pathogenic T cell response. In furtherembodiments, the subject is one that is suffering from or at risk ofsuffering from an autoimmune disease or an auto-inflammatory disease. Ina specific embodiment, the autoimmune disease or auto-inflammatorydisease is selected from rheumatoid arthritis, allergy, asthma, systemiconset juvenile arthritis, inflammatory bowel disease, systemic lupuserythematosus, multiple sclerosis, type 1 diabetes, graft rejection,graft versus host disease, colitis, and Crohn's disease.

In some embodiments, the subject is one that will receive or hasreceived transplanted tissues. In a related embodiment, the transplantedtissue is an allograft. An allograft (also known as allotransplantation,allogeneic transplant, or homograft) is the transplantation of cells,tissues, or organs, to a recipient from a genetically non-identicaldonor of the same species. In a related embodiment, the subject is onethat has a complication from the transplanted tissue, wherein thecomplication is graft rejection or GVHD.

In some embodiments, the APC-targeted antibody is administered prior totissue transplantation. When the antibody or antigen binding fragmentthereof is administered prior to tissue transplantation, the method mayfurther comprise the prevention of a complication relating to thetransplanted tissue, wherein the complication comprises GVHD or graftrejection.

In some embodiments, the APC-targeted antibody is administered aftertissue transplantation. When the antibody or antigen binding fragmentthereof is administered after tissue transplantation, the method mayfurther comprise treating a complication from the transplanted tissue,wherein the complication comprises GVHD or graft rejection.

The tissue used in transplantation may be any tissue known in the art tobe therapeutically useful for transplantation. Non-limiting examples oftissue transplantations include anterior cruciate ligament (ACL); jointreconstruction in the knee and ankle; meniscal replacement;reconstruction due to cancer or trauma; ridge augmentation in dentalprocedures; shoulder repair; spinal fusion; urological tissues; skintransplants; corneal transplants; heart transplants; heart valves; lungtransplantation; intestinal transplantation such as isolated smallbowel, intestine, or multivisceral; liver transplants; kidneytransplants; bone marrow transplants; bone allograft; and ligament ortendon allograft.

In one embodiment, the transplanted tissue comprises immune cells. Theterm immune cells includes cells of the immune system that are involvedin defending the body against both infectious disease and foreignmaterials. Immune cells may include, for example, neutorphils,eosinophils, basophils, lymphocytes such as b cells and t cells, andmonocytes. T cells may include, for example, CD4+, CD8+, T helper cells,cytotoxic T cells, γδ T cells, regulatory T cells, suppressor T cells,and natural killer cells.

In another embodiment, the transplanted tissue comprises stem cells.Stem cell types are known in the art. Non-limiting examples of stemcells include hematopoietic stem cells, neural stem cells, and embryonicstem cells. In one embodiment, the stem cells are hematopoietic stemcells. In a further embodiment, the transplanted tissue comprises bonemarrow. In a yet further embodiment, the transplanted tissue comprisesblood. In another embodiment, the transplanted tissue comprises skincells.

In some embodiments, the APC-targeted antibody operatively linked toIL-10 or a fragment thereof is administered in an amount effect for themaintenance of pathogen-specific immunity in the subject.

The IL-10 polypeptide may be a polypeptide or fragment of an IL-10protein known in the art or described herein by accession numberNP_000563.1. In some embodiments, the IL-10 polypeptide comprises SEQ IDNO:5. In some embodiments, IL-10 is covalently linked to the antibody.In some embodiments, the covalent linkage is through a peptide bond. TheIL-10 polypeptide may also be linked to the antibody through bindingpolypeptides. In one embodiment, the binding polypeptides are dockerinand cohesin.

In some embodiments, the method further comprises administration of anantigen or allergen. The antigen or allergen may be operatively linkedto the APC-targeted antibody or to IL-10. In some embodiments, theantigen or allergen is covalently linked (i.e. by a peptide bond) to theAPC-targeted antibody, antigen binding fragment thereof, or IL-10. Whenthe antigen or allergen is operatively linked to the APC-targetedantibody, antigen binding fragment thereof, or IL-10, it may be linkedthrough binding polypeptides. Binding peptides include, for example,dockerin and cohesin.

In further embodiments, the compositions or methods do not comprise anantigen or allergen or the administration of an allergy or antigen. Forexample, an antigen or allergen is not operatively (either directly orindirectly) linked to the APC-targeted antibody. In some embodiments,the compositions consists essentially of an antigen presenting cell(APC)-targeted antibody operatively linked to IL-10 or a fragmentthereof.

In further embodiments, the compositions or methods do not comprise aTLR molecule or the administration of a TLR molecule.

In some embodiments, the antibody may comprise a γ4 constant region. Ina related embodiment, the γ4 constant region comprises a substitution ofglutamic acid for leucine at residue 235. In another embodiment, γ4constant region comprises a substitution of proline for serine atresidue 228 in the hinge region.

In certain embodiments, the methods comprises multiple administrationsof the composition. The administrations may be days, weeks, months,years, or decades apart. The compositions comprising the conjugatedescribed herein may be administered orally, intravenously,subcutaneously, intradermally, intramuscularly, intranasally, byinjection, by inhalation, mucosally, and/or by using a nebulizer.

In certain embodiments of the methods described herein, theanti-DC-ASGPR antibody or antigen binding fragment is administered in atherapeutically effective amount. In certain embodiments, the antibodyor antigen binding fragment is administered in an amount that increasesproduction of IL-10 in the subject. In a further embodiment, theantibody or antigen binding fragment is administered in an amountwhereby the subject maintains pathogen-specific immunity afteradministration of the antibody or antigen binding fragment.

In some embodiments, the anti-DC-ASGPR antibody or antigen bindingfragment thereof may be administered in a pharmaceutical composition. Insome embodiments, the pharmaceutical composition does not contain anantigen or does not contain detectable amounts of an antigen. In afurther embodiment, the pharmaceutical composition consists essentiallyof an anti-DC-ASGPR antibody. In further embodiments, the antibody orantigen binding fragment thereof is not conjugated to an antigen or isnot is not conjugated to a dockerin or cohesion molecule. In yet furtherembodiments, the antibody is not covalently or operatively linked to anantigen.

The term “operatively linked” refers to a situation where two componentsare combined to form the active complex prior to binding at the targetsite. For example, an antibody conjugated to one-half of acohesion-docerin complex and an antigen complexed to the other one-halfof the cohesion-docerin complex are operatively linked throughcomplexation of the cohesion and docerin molecules.

Also disclosed herein are compositions comprising the antibodies andantibody conjugates as described herein.

Aspects of the disclosure relate to APC-targeted antibodies andAPC-targeted antibodies conjugated to IL-10 in pharmaceuticalcompositions and for use in the preparation of medicaments for treatingan autoimmune and/or inflammatory condition described herein.

Aspects also relate to an APC-targeted antibody or an APC-targetedantibody conjugated to IL-10 in pharmaceutical compositions and for usein the preparation of medicaments for inducing immune tolerance orsuppressing a T cell response in a subject having or at risk ofdeveloping an autoimmune or inflammatory response, wherein theautoimmune or inflammatory response is caused by an autoimmune orinflammatory disease described herein.

As used herein the specification, “a” or “an” may mean one or more. Asused herein in the claim(s), when used in conjunction with the word“comprising”, the words “a” or “an” may mean one or more than one. Acomposition with the words “consisting essentially of” is intended toexclude any active ingredients not specifically recited in thecomposition. Examples of active ingredients include cytokines, TLRs,antigens, adjuvants, etc. . . . . In any of the embodiments describedherein, embodiments consisting essentially of the recited elements isalso contemplated.

The use of the term “or” in the claims is used to mean “and/or” unlessexplicitly indicated to refer to alternatives only or the alternativesare mutually exclusive, although the disclosure supports a definitionthat refers to only alternatives and “and/or.” As used herein “another”may mean at least a second or more.

Throughout this application, the term “about” is used to indicate that avalue includes the inherent variation of error for the device, themethod being employed to determine the value, or the variation thatexists among the study subjects.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1 shows that recombinant fusion proteins of antibody and IL-10target human APCs.

FIG. 2 shows that antibody-IL-10 fusion proteins inhibit DC maturationinduced by E. coli lipopolysaccharide.

FIG. 3 demonstrates the dose sparing effects of the targeted IL-10fustion proteins.

FIG. 4 shows that treatment of PBMCs from healthy donors withanti-DC-ASGPR mAb reduces the proliferation of CD4⁺ and CD8⁺ T cellsfrom MHC-mismatched donors. Summary of data generated with PBMCs from 6pairs of MHC-mismatched healthy donors (Mean±SD).

FIG. 5 shows that blocking IL-10 partially recovers allogeneic CD4+ Tcells proliferation induced by anti-DC-ASGPR-activated PBMCs.

FIG. 6 shows that allogeneic CD4+ T cells cultured withanti-DC-ASGPR-activated PBMCs secrete decreased IFNγ but increased IL-10during restimulation.

FIG. 7 demonstrates the effect of anti-DC-ASGPR mAb on delay ofxenogenic GVHD in NOG mice.

FIG. 8 shows the hypothetical pathways of DC-ASGPR-induced suppressionof allogeneic T cell responses.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

Methods and compositions described herein can be used to treat orprevent inflammatory and/or autoimmune disorders or for inducing immunetolerance. It was discovered that delivering the anti-inflammatorycytokine, IL-10, to human antigen presenting cells (APCs) can suppressand alter the pathophysiologic functions of APCs in the patients. It iscontemplated that targeted delivery of anti-inflammatory cytokines tothe APCs in the patients is expected to result in more effective andpro-longed immune tolerance in the patients. Delivering IL-10 to APCscan directly suppress ongoing inflammatory reaction in a short termperiod and can also induce regulatory T cells which can prolong theeffectiveness of the treatment. Furthermore, the methods describedherein provide a dose sparing effect such that the targeted delivery ofIL-10 requires a smaller amount or dose to achieve the same effect as anon-targeted IL-10.

I. ANTIBODIES

Methods and compositions of the disclosure relate to APC-targetedantibodies and antibody binding fragments thereof. In some embodiments,the antibodies are operatively linked to IL-10. As used herein, an“antibody” includes whole antibodies and any antigen binding fragment ora single chain thereof. Thus the term “antibody” includes any protein orpeptide-containing molecule that comprises at least a portion of animmunoglobulin molecule. Examples of such include, but are not limitedto a complementarity determining region (CDR) of a heavy or light chainor a ligand binding portion thereof, a heavy chain or light chainvariable region, a heavy chain or light chain constant region, aframework (FR) region or any portion thereof or at least one portion ofa binding protein.

The antibody can be any of the various antibodies described herein,non-limiting, examples of such include a polyclonal antibody, amonoclonal antibody, a chimeric antibody, a recombinant antibody, ahuman antibody, a veneered antibody, a diabody, a humanized antibody, anantibody derivative, a recombinant humanized antibody, or a derivativeor fragment of each thereof.

Antibodies can be generated using conventional techniques known in theart and are well-described in the literature. Several methodologiesexist for production of polyclonal antibodies. For example, polyclonalantibodies are typically produced by immunization of a suitable mammalsuch as, but not limited to, chickens, goats, guinea pigs, hamsters,horses, mice, rats, and rabbits. An antigen is injected into the mammal,induces the B-lymphocytes to produce immunoglobulins specific for theantigen. Immunoglobulins may be purified from the mammal's serum. Commonvariations of this methodology include modification of adjuvants, routesand site of administration, injection volumes per site and the number ofsites per animal for optimal production and humane treatment of theanimal. For example, adjuvants typically are used to improve or enhancean immune response to antigens. Most adjuvants provide for an injectionsite antigen depot, which allows for a stow release of antigen intodraining lymph nodes. Other adjuvants include surfactants which promoteconcentration of protein antigen molecules over a large surface area andimmunostimulatory molecules. Non-limiting examples of adjuvants forpolyclonal antibody generation include Freund's adjuvants, Ribi adjuvantsystem, and Titermax. Polyclonal antibodies can be generated usingmethods known in the art some of which are described in U.S. Pat. Nos.7,279,559; 7,119,179; 7,060,800; 6,709,659; 6,656,746; 6,322,788;5,686,073; and 5,670,153.

Unless specified otherwise, the antibodies can be polyclonal ormonoclonal and can be isolated from any suitable biological source,e.g., murine, rat, sheep or canine.

In a specific embodiment, the antibody is a monoclonal antibody. As usedherein, “monoclonal antibody” refers to an antibody obtained from asubstantially homogeneous antibody population. Monoclonal antibodies arehighly specific, as each monoclonal antibody is directed against asingle determinant on the antigen. The antibodies may be detectablylabeled, e.g., with a radioisotope, an enzyme which generates adetectable product, a fluorescent protein, and the like. The antibodiesmay be further conjugated to other moieties, such as members of specificbinding pairs, e.g., biotin (member of biotin-avidin specific bindingpair), and the like. The antibodies may also be bound to a solidsupport, including, but not limited to, polystyrene plates or beads, andthe like.

Monoclonal antibodies can be generated using conventional hybridomatechniques known in the art and well-described in the literature. Forexample, a hybridoma is produced by fusing a suitable immortal cell line(e.g., a myeloma cell line such as, but not limited to, Sp2/0,Sp2/0-AG14, NSO, NS1, NS2, AE-1, L.5, P3X63Ag8,653, Sp2 SA3, Sp2 MAI,Sp2 SS1, Sp2 SA5, U397, MIA 144, ACT IV, MOLT4, DA-1, JURKAT, WEHI,K-562, COS, RAJI, NIH 313, HL-60, MLA 144, NAMAIWA, NEURO 2A, CHO,PerC.6, YB2/O) or the like, or heteromyelomas, fusion products thereof,or any cell or fusion cell derived there from, or any other suitablecell line as known in the art, with antibody producing cells, such as,but not limited to, isolated or cloned spleen, peripheral blood, lymph,tonsil, or other immune or B cell containing cells, or any other cellsexpressing heavy or light chain constant or variable or framework or CDRsequences, either as endogenous or heterologous nucleic acid, asrecombinant or endogenous, viral, bacterial, algal, prokaryotic,amphibian, insect, reptilian, fish, mammalian, rodent, equine, ovine,goat, sheep, primate, eukaryotic, genomic DNA, cDNA, rDNA, mitochondrialDNA or RNA, chloroplast DNA or RNA, hnRNA, mRNA, tRNA, single, double ortriple stranded, hybridized, and the like or any combination thereof.Antibody producing cells can also be obtained from the peripheral bloodor, preferably the spleen or lymph nodes, of humans or other suitableanimals that have been immunized with the antigen of interest. Any othersuitable host cell can also be used for expressing-heterologous orendogenous nucleic acid encoding an antibody, specified fragment orvariant thereof. The fused cells (hybridomas) or recombinant cells canbe isolated using selective culture conditions or other suitable knownmethods, and cloned by limiting dilution or cell sorting, or other knownmethods.

Other suitable methods of producing or isolating antibodies of therequisite specificity can be used, including, but not limited to,methods that select recombinant antibody from a peptide or proteinlibrary (e.g., but not limited to, a bacteriophage, ribosome,oligonucleotide, cDNA, or the like, display library; e.g., as availablefrom various commercial vendors such as MorphoSys (Martinsreid/Planegg,Del.), BioInvent (Lund, Sweden), Affitech (Oslo, Norway) using methodsknown in the art. Art known methods are described in the patentliterature some of which include U.S. Pat. Nos. 4,704,692; 5,723,323;5,763,192; 5,814,476; 5,817,483; 5,824,514; 5,976,862. Alternativemethods rely upon immunization of transgenic animals (e.g., SCID mice,Nguyen et al. (1977) Microbiol. Immunol. 41:901-907 (1997); Sandhu etal. (1996) Crit, Rev. Biotechnol. 16:95-118; Eren et al. (1998) Mumma93:154-161 that are capable of producing a repertoire of humanantibodies, as known in the art and/or as described herein. Suchtechniques, include, but are not limited to, ribosome display Wanes etal. (1997) Proc. Natl. Acad. Sci. USA, 94:4937-4942; Hanes et al, (1998)Proc. Natl. Acad. Sci. USA 95:14130-14135); single cell antibodyproducing technologies (e.g., selected lymphocyte antibody method(“SLAM”) (U.S. Pat. No. 5,627,052, Wen et al, (1987) J. Immunol17:887-892; Babcook et al. (1996) Proc. Natl. Acad. Sci. USA93:7843-7848); gel microdroplet and flow cytometry (Powell et al. (1990)Biotechnol. 8:333-337; One Cell Systems, (Cambridge, Mass.); Gray et al.(1995) J. Imm. Meth. 182:155-163; and Kenny et al, (1995) Bio. Technol.13:787-790); B-cell selection (Steenbakkers et al. (1994) Molec. Biol.Reports 19:125-134).

The terms “polyclonal antibody” or “polyclonal antibody composition” asused herein refer to a preparation of antibodies that are derived fromdifferent B-cell lines. They are a mixture of immunoglobulin moleculessecreted against a specific antigen, each recognizing a differentepitope.

The term “mouse antibody” as used herein, is intended to includeantibodies having variable and constant regions derived from mousegermline immunoglobulin sequences.

As used herein, chimeric antibodies are antibodies whose light and heavychain genes have been constructed, typically by genetic engineering,from antibody variable and constant region genes belonging to differentspecies. In one embodiment, the antibody is a mouse/human chimericantibody.

In further embodiments, the antibody comprises a modification and is an“antibody derivative.” The term “antibody derivative” includespost-translational modification to linear polypeptide sequence of theantibody or fragment. For example, U.S. Pat. No. 6,602,684 B1 describesa method for the generation of modified glycol-forms of antibodies,including whole antibody molecules, antibody fragments, or fusionproteins that include a region equivalent to the Fc region of animmunoglobulin, having enhanced Fe-mediated cellular toxicity, andglycoproteins so generated.

The antibodies provided herein also include derivatives that aremodified by the covalent attachment of any type of molecule to theantibody such that covalent attachment does not prevent the antibodyfrom generating an anti-idiotypic response. Antibody derivativesinclude, but are not limited to, antibodies that have been modified byglycosylation, acetylation, pegylation, phosphorylation, amidation,derivatization by known protecting/blocking groups, proteolyticcleavage, linkage to a cellular ligand or other protein, etc.Additionally, the derivatives may contain one or more non-classicalamino acids.

Antibody derivatives can also be prepared by delivering a polynucleotideencoding an antibody to a suitable host such as to provide transgenicanimals or mammals, such as goats, cows, horses, sheep, and the like,that produce such antibodies in their milk. These methods are known inthe art and are described for example in U.S. Pat. Nos. 5,827,690;5,849,992; 4,873,316; 5,849,992; 5,994,616; 5,565,362; and 5,304,489.

Antibody derivatives also can be prepared by delivering a polynucleotideto provide transgenic plants and cultured plant cells (e.g., but notlimited to tobacco, maize, and duckweed) that produce such antibodies,specified portions or variants in the plant parts or in cells culturedtherefrom. Antibody derivatives have also been produced in large amountsfrom transgenic plant seeds including antibody fragments, such as singlechain antibodies (scFv's), including tobacco seeds and potato tubers.See, e.g., Conrad et al. (1998) Plant Mol. Biol. 38:101-109 andreferences cited therein. Thus, antibodies can also be produced usingtransgenic plants, according to know methods.

Antibody derivatives also can be produced, for example, by addingexogenous sequences to modify immunogenicity or reduce, enhance ormodify binding, affinity, on-rate, off-rate, avidity, specificity,half-life, or any other suitable characteristic. Generally part or allof the non-human or human CDR sequences are maintained while thenon-human sequences of the variable and constant regions are replacedwith human or other amino acids.

The term “variable region” refers to a portion of the antibody thatgives the antibody its specificity for binding antigen. The variableregion is typically located at the ends of the heavy and light chains.Variable loops of β-strands, three each on the light (VL) and heavy (VH)chains are responsible for binding to the antigen. These loops arereferred to as the “complementarity determining regions” (CDRs).

In general, the CDR residues are directly and most substantiallyinvolved in influencing antigen binding. Humanization or engineering ofantibodies can be performed using any known method such as, but notlimited to, those described in U.S. Pat. Nos. 5,723,323; 5,976,862;5,824,514; 5,817,483; 5,814,476; 5,763,192; 5,723,323; 5,766,886;5,714,352; 6,204,023; 6,180,370; 5,693,762; 5,530,101; 5,585,089;5,225,539; and 4,816,567.

The term “constant region” refers to a portion of the antibody that isidentical in all antibodies of the same isotype. The constant regiondiffers in antibodies of different isotypes.

In one embodiment, the antibody is a humanized antibody. As used herein,the term “humanized antibody” or “humanized immunoglobulin” refers to ahuman/non-human chimeric antibody that contains a minimal sequencederived from non-human immunoglobulin. For the most part, humanizedantibodies are human immunoglobulins (recipient antibody) in whichresidues from a variable region of the recipient are replaced byresidues from a variable region of a non-human species (donor antibody)such as mouse, rat, rabbit, or non-human primate having the desiredspecificity, affinity and capacity. Humanized antibodies may compriseresidues that are not found in the recipient antibody or in the donorantibody. The humanized antibody can optionally also comprise at least aportion of an immunoglobulin constant region (Fc), typically that of ahuman immunoglobulin, a non-human antibody containing one or more aminoacids in a framework region, a constant region or a CDR, that have beensubstituted with a correspondingly positioned amino acid from a humanantibody. In general, humanized antibodies are expected to produce areduced immune response in a human host, as compared to a non-humanizedversion of the same antibody. The humanized antibodies may haveconservative amino acid substitutions which have substantially no effecton antigen binding or other antibody functions. Conservativesubstitutions groupings include: glycine-alanine,valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine,alanine-valine, serine-threonine and asparagine-glutamine.

Chimeric, humanized or primatized antibodies can be prepared based onthe sequence of a reference monoclonal antibody prepared using standardmolecular biology techniques. DNA encoding the heavy and light chainimmunoglobulins can be obtained from the hybridoma of interest andengineered to contain non-reference (e.g., human) immunoglobulinsequences using standard molecular biology techniques. For example, tocreate a chimeric antibody, the murine variable regions can be linked tohuman constant regions using methods known in the art (U.S. Pat. No.4,816,567). To create a humanized antibody, the murine CDR regions canbe inserted into a human framework using methods known in the art (U.S.Pat. Nos. 5,225,539 and 5,530,101; 5,585,089; 5,693,762 and 6,180,370).Similarly, to create a primatized antibody the murine CDR regions can beinserted into a primate framework using methods known in the art (WO93/02108 and WO 99/55369). Methods of determining CDRs from the sequenceof a variable region are known in the art (see, for example, Zhao andLu, “A germline knowledge based computational approach for determiningantibody complementarity determining regions.” Mol. Immunol., (2010)47(4):694-700, which is herein incorporated by reference).

Techniques for making partially to fully human antibodies are known inthe art and any such techniques can be used. According to oneembodiment, fully human antibody sequences are made in a transgenicmouse which has been engineered to express human heavy and light chainantibody genes. Multiple strains of such transgenic mice have been madewhich can produce different classes of antibodies. B cells fromtransgenic mice which are producing a desirable antibody can be fused tomake hybridoma cell lines for continuous production of the desiredantibody. (See for example, Russel et al. (2000) Infection and ImmunityApril 2000:1820-1826; Gallo et al. (2000) European J. of Immun.30:534-540; Green (1999) J. of Immun. Methods 231:11-23; Yang et al.(1999A) J. of Leukocyte Biology 66:401-410; Yang (1999B) Cancer Research59(6):1236-1243; Jakobovits (1998) Advanced Drug Reviews 31:33-42; Greenand Jakobovits (1998) J. Exp. Med. 188(3):483-495; Jakobovits (1998)Exp. Opin. Invest. Drugs 7(4):607-614; Tsuda et al. (1997) Genomics42:413-421; Sherman-Gold (1997) Genetic Engineering News 17(14); Mendezet al. (1997) Nature Genetics 15:146-156; Jakobovits (1996) Weir'sHandbook of Experimental Immunology, The Integrated Immune System Vol.IV, 194.1-194.7; Jakobovits (1995) Current Opinion in Biotechnology6:561-566; Mendez et al, (1995) Genomics 26:294-307; Jakobovits (1994)Current Biology 4(8):761-763; Arbones et al. (1994):Immunity1(4):247-260; Jakobovits (1993) Nature 362(6417):255-258; Jakobovits etal. (1993) Proc. Natl. Acad. Sci. USA 90(6):2551-2555; and U.S. Pat. No.6,075,181.)

Antibodies also can be modified to create chimeric antibodies. Chimericantibodies are those in which the various domains of the antibodies'heavy and light chains are coded for by DNA from more than one species.See, e.g., U.S. Pat. No. 4,816,567.

Alternatively, antibodies can also be modified to create veneeredantibodies. Veneered antibodies are those in which the exterior aminoacid residues of the antibody of one species are judiciously replaced or“veneered” with those of a second species so that the antibodies of thefirst species will not be immunogenic in the second species therebyreducing the immunogenicity of the antibody. Since the antigenicity of aprotein is primarily dependent on the nature of its surface, theimmunogenicity of an antibody could be reduced by replacing the exposedresidues which differ from those usually found in another mammalianspecies antibodies. This judicious replacement of exterior residuesshould have little, or no, effect on the interior domains, or on theinterdomain contacts. Thus, ligand binding properties should beunaffected as a consequence of alterations which are limited to thevariable region framework residues. The process is referred to as“veneering” since only the outer surface or skin of the antibody isaltered, the supporting residues remain undisturbed.

The procedure for “veneering” makes use of the available sequence datafor human antibody variable domains compiled by Kabat et al. (1987)Sequences of Proteins of Immunological interest, 4th ed., Bethesda, Md.,National Institutes of Health, updates to this database, and otheraccessible U.S. and foreign databases (both nucleic acid and protein).Non-limiting examples of the methods used to generate veneeredantibodies include EP 519596; U.S. Pat. No. 6,797,492; and described inPadlan et al. (1991) Mol. Immunol. 28(4-5):489-498.

The term “antibody derivative” also includes “diabodies” which are smallantibody fragments with two antigen-binding sites, wherein fragmentscomprise a heavy chain variable domain (VH) connected to a light chainvariable domain (VL) in the same polypeptide chain. (See for example, EP404,097; WO 93/11161; and Hollinger et al. (1993) Proc. Natl. Acad. Sci.USA 90:6444-6448.) By using a linker that is too short to allow pairingbetween the two domains on the same chain, the domains are forced topair with the complementary domains of another chain and create twoantigen-binding sites. (See also, U.S. Pat. No. 6,632,926 to Chen et al,which discloses antibody variants that have one or more amino acidsinserted into a hypervariable region of the parent antibody and abinding affinity for a target antigen which is at least about two foldstronger than the binding affinity of the parent antibody for theantigen).

The term “antibody derivative” further includes engineered antibodymolecules, fragments and single domains such as scFv, dAbs, nanobodies,minibodies, Unibodies, and Affibodies & Hudson (2005) Nature Biotech23(9):1126-36; U.S. Patent Publication US 2006/0211088; PCT PublicationWO2007/059782; U.S. Pat. No. 5,831,012).

The term “antibody derivative” further includes “linear antibodies”. Theprocedure for making linear antibodies is known in the art and describedin Zapata et al. (1995) Protein Eng. 8(10):1057-1062. Briefly, theseantibodies comprise a pair of tandem Ed segments (V.sub.H-C.sub.H1-VH-C.sub.H1) which form a pair of antigen binding regions. Linearantibodies can be bispecific or monospecific.

Antibodies can be recovered and purified from recombinant cell culturesby known methods including, but not limited to, protein A purification,ammonium sulfate or ethanol precipitation, acid extraction, anion orcation exchange chromatography, phosphocellulose chromatography,hydrophobic interaction chromatography, affinity chromatography,hydroxylapatite chromatography and lectin chromatography. Highperformance liquid chromatography (“HPLC”) can also be used forpurification.

It also is possible to determine without undue experimentation, whetheran antibody has the same specificity as antibodies contemplated hereinby determining whether the antibody being tested prevents an antibodyfrom binding the protein or polypeptide with which the antibody isnormally reactive. If the antibody being tested competes with anantibody used in embodiments described herein as shown by a decrease inbinding by the monoclonal antibody, then it is likely that the twoantibodies bind to the same or a closely related epitope. Alternatively,one can pre-incubate an antibody for use in embodiments with a proteinwith which it is normally reactive, and determine if the antibody beingtested is inhibited in its ability to bind the antigen. If the antibodybeing tested is inhibited then, in all likelihood, it has the same, or aclosely related, epitopic specificity as the antibody for use inembodiments described herein.

The term “antibody” also is intended to include antibodies of allimmunoglobulin isotypes and subclasses unless specified otherwise. Anisotype refers to the genetic variations or differences in the constantregions of the heavy and light chains of an antibody. In humans, thereare five heavy chain isotypes: IgA, IgD, IgG, IgE, and IgM and two lightchain isotypes: kappa and lambda. The IgG class is divided into fourisotypes: IgG1, IgG2, IgG3 and IgG4 in humans, and IgG1, IgG2a, IgG2band IgG3 in mice. They share more than 95% homology in the amino acidsequences of the Fc regions but show major differences in the amino acidcomposition and structure of the hinge region. Particular isotypes of amonoclonal antibody can be prepared either directly by selecting from aninitial fusion, or prepared secondarily, from a parental hybridomasecreting a monoclonal antibody of different isotype by using the sibselection technique to isolate class switch variants using the proceduredescribed in Steplewski et al. (1985) Proc. Natl. Acad. Sci. USA 82:8653or Spira et al, (1984) J. Immunol. Methods 74:307. Alternatively,recombinant DNA techniques may be used.

The isolation of other monoclonal antibodies with the specificity of themonoclonal antibodies described herein can also be accomplished by oneof ordinary skill in the art by producing anti-idiotypic antibodies.Herlyn et al. (1986) Science 232:100. An anti-idiotypic antibody is anantibody which recognizes unique determinants present on the monoclonalantibody of interest.

In some aspects, it will be useful to detectably or therapeuticallylabel the antibody. Methods for conjugating antibodies to these agentsare known in the art. For the purpose of illustration only, antibodiescan be labeled with a detectable moiety such as a radioactive atom, achromophore, a fluorophore, or the like. Such labeled antibodies can beused for diagnostic techniques, either in vivo, or in an isolated testsample.

In certain embodiments, the antibody or antigen binding fragment furthercomprises a modification. The modification may be a conservative aminoacid mutation within the VH and/or VL CDR 1, CDR 2 and/or CDR 3 regions,of conservative amino acid mutations in the Fc hinge region, pegylation,conjugation to a serum protein, conjugation to human serum albumin,conjugation to a detectable label, conjugation to a diagnostic agent,conjugation to an enzyme, conjugation to a fluorescent, luminescent, orbioluminescent material, conjugation to a radioactive material, orconjugation to a therapeutic agent.

As used herein, the term “label” intends a directly or indirectlydetectable compound or composition that is conjugated directly orindirectly to the composition to be detected, e.g., polynucleotide orprotein such as an antibody so as to generate a “labeled” composition.The term also includes sequences conjugated to the polynucleotide thatwill provide a signal upon expression of the inserted sequences, such asgreen fluorescent protein (GFP) and the like. The label may bedetectable by itself (e.g. radioisotope labels or fluorescent labels)or, in the case of an enzymatic label, may catalyze chemical alterationof a substrate compound or composition which is detectable. The labelscan be suitable for small scale detection or more suitable forhigh-throughput screening. As such, suitable labels include, but are notlimited to radioisotopes, fluorochromes, chemiluminescent compounds,dyes, and proteins, including enzymes. The label may be simply detectedor it may be quantified. A response that is simply detected generallycomprises a response whose existence merely is confirmed, whereas aresponse that is quantified generally comprises a response having aquantifiable (e.g., numerically reportable) value such as an intensity,polarization, and/or other property. In luminescence or fluorescenceassays, the detectable response may be generated directly using aluminophore or fluorophore associated with an assay component actuallyinvolved in binding, or indirectly using a luminophore or fluorophoreassociated with another (e.g., reporter or indicator) component.

Examples of luminescent labels that produce signals include, but are notlimited to bioluminescence and chemiluminescence. Detectableluminescence response generally comprises a change in, or an occurrenceof, a luminescence signal. Suitable methods and luminophores forluminescently labeling assay components are known in the art anddescribed for example in Haugland, Richard P. (1996) Handbook ofFluorescent Probes and Research Chemicals (6.sup.th ed.). Examples ofluminescent probes include, but are not limited to, aequorin andluciferases.

Examples of suitable fluorescent labels include, but are not limited to,fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosin,coumarin, methyl-coumarins, pyrene, Malacite green, stilbene, LuciferYellow, Cascade Blue™, and Texas Red. Other suitable optical dyes aredescribed in the Haugland, Richard P. (1996) Handbook of FluorescentProbes and Research Chemicals (6.sup.th ed.).

In another aspect, the fluorescent label is functionalized to facilitatecovalent attachment to a cellular component present in or on the surfaceof the cell or tissue such as a cell surface marker. Suitable functionalgroups, including, but not are limited to, isothiocyanate groups, aminogroups, haloacetyl groups, maleimides, succinimidyl esters, and sulfonylhalides, all of which may be used to attach the fluorescent label to asecond molecule. The choice of the functional group of the fluorescentlabel will depend on the site of attachment to either a linker, theagent, the marker, or the second labeling agent.

Attachment of the fluorescent label may be either directly to thecellular component or compound or alternatively, can by via a linker.Suitable binding pairs for use in indirectly linking the fluorescentlabel to the intermediate include, but are not limited to,antigens/antibodies, e.g., rhodamine/anti-rhodamine, biotin/avidin andbiotin/strepavidin.

The coupling of antibodies to low molecular weight haptens can increasethe sensitivity of the antibody in an assay. The haptens can then bespecifically detected by means of a second reaction. For example, it iscommon to use haptens such as biotin, which reacts avidin, ordinitrophenol, pyridoxal, and fluorescein, which can react with specificanti-hapten antibodies. See, Harlow and Lane (1988) supra.

The variable region of an antibody can be modified by mutating aminoacid residues within the VH and/or VL CDR 1, CDR 2 and/or CDR 3 regionsto improve one or more binding properties (e.g., affinity) of theantibody. Mutations may be introduced by site-directed mutagenesis orPCR-mediated mutagenesis and the effect on antibody binding, or otherfunctional property of interest, can be evaluated in appropriate invitro or in vivo assays. Preferably conservative modifications areintroduced and typically no more than one, two, three, four or fiveresidues within a CDR region are altered. The mutations may be aminoacid substitutions, additions or deletions.

Framework modifications can be made to the antibodies to decreaseimmunogenicity, for example, by “backmutating” one or more frameworkresidues to the corresponding germline sequence.

In addition, an antibody may be engineered to include modificationswithin the Fc region to alter one or more functional properties of theantibody, such as serum half-fife, complement fixation, Fc receptorbinding, and/or antigen-dependent cellular cytotoxicity. Suchmodifications include, but are not limited to, alterations of the numberof cysteine residues in the hinge region to facilitate assembly of thelight and heavy chains or to increase or decrease the stability of theantibody (U.S. Pat. No. 5,677,425) and amino acid mutations in the Fchinge region to decrease die biological half life of the antibody (U.S.Pat. No. 6,165,745).

Additionally, one or more antibodies may be chemically modified.Glycosylation of an antibody can be altered, for example, by modifyingone or more sites of glycosylation within the antibody sequence toincrease the affinity of the antibody for antigen (U.S. Pat. Nos.5,714,350 and 6,350,861). Alternatively, to increase antibody-dependentcell-mediated cytotoxicity, a hypofucosylated antibody having reducedamounts of fucosyl residues or an antibody having increased bisectingGlcNac structures can be obtained by expressing the antibody in a hostcell.sub.—with altered glycosylation mechanism (Shields, R. L. et al.,2002 J. Biol. Chem. 277:26733-26740; Umana et al., 1999 Nat. Biotech.17:176-180).

Antibodies can be pegylated to increase biological half-life by reactingthe antibody or fragment thereof with polyethylene glycol (PEG) or areactive ester or aldehyde derivative of PEG, under conditions in whichone or more PEG groups become attached to the antibody or antibodyfragment. Antibody pegylation may be carried out by an acylationreaction or an alkylation reaction with a reactive PEG molecule (or ananalogous reactive watersoluble polymer). As used herein, the term“polyethylene glycol” is intended to encompass any of the forms of PEGthat have been used to derivatize other proteins, such as mono (C1-C10)alkoxy- or aryloxy-polyethylene glycol or polyethylene glycol-maleimide.The antibody to be pegylated can be an aglycosylated antibody. Methodsfor pegylating proteins are known in the art and can be applied to oneor more antibodies (EP 0 154 316 and EP 0 401 384).

Additionally, antibodies may be chemically modified by conjugating orfusing the antigen-binding region of the antibody to serum protein, suchas human serum albumin, to increase half-life of the resulting molecule.Such approach is for example described in EP 0322094 and EP 0 486 525.

The antibodies or fragments thereof may be conjugated to a diagnosticagent and used diagnostically, for example, to monitor the developmentor progression of a disease and determine the efficacy of a giventreatment regimen. Examples of diagnostic agents include enzymes,prosthetic groups, fluorescent materials, luminescent materials,bioluminescent materials, radioactive materials, positron emittingmetals using various positron emission tomographies, and nonradioactiveparamagnetic metal ions. The detectable substance may be coupled orconjugated either directly to the antibody or fragment thereof, orindirectly, through a 1 inker using techniques known in the art.Examples of suitable enzymes include horseradish peroxidase, alkalinephosphatase, beta-galactosidase, or acetylcholinesterase. Examples ofsuitable prosthetic group complexes include streptavidin/biotin andavidin/biotin. Examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin. Anexample of a luminescent material includes luminol. Examples ofbioluminescent materials include luciferase, luciferin, and aequorin.Examples of suitable radioactive material include.sup.125I, .sup.131I,Indium-111, Lutetium-171, Bismuth-212, Bismuth-213, Astatine-211,Copper-62, Copper-64, Copper-67, Yttrium-90, Iodine-125, Iodine-131,Phosphorus-32, Phosphorus-33, Scandium-47, Silver-111, Gallium-67,Praseodymium-142, Samarium-153, Terbium-161, Dysprosium-166,Holmium-166, Rhenium-186, Ithenium-188, Rhenium-189, Lead-212,Radium-223, Actinium-225, Iron-59, Selenium-75, Arsenic-77,Strontium-89, Molybdenum-99, Rhodium-1105, Palladium-109,Praseodymium-143, Promethium-149, Erbium-169, Iridium-194, Gold-198,Gold-199, and Lead-211. Monoclonal antibodies may be indirectlyconjugated with radiometal ions through the use of bifunctionalchelating agents that are covalently linked to the antibodies. Chelatingagents may be attached through amities (Meares et al., 1984 Anal.Biochem. 142: 68-78); sulfhydral groups (Koyama 1994 Chem. Abstr. 120:217262t) of amino acid residues and carbohydrate groups (Rodwell et al.1986 PNAS USA 83: 2632-2636; Quadri et al. 1993 Nucl. Med. Biol. 20:559-570).

Additional suitable conjugated molecules include ribonuclease (RNase),DNase I, an antisense nucleic acid, an inhibitory RNA molecule such as asiRNA molecule, an immunostimulatory nucleic acid, aptamers, ribozymes,triplex forming molecules, and external guide sequences. Aptamers aresmall nucleic acids ranging from 15-50 bases in length that fold intodefined secondary and tertiary structures, such as stern-loops orG-quartets, and can bind small molecules, such as ATP (U.S. Pat. No.5,631,146) and theophiline (U.S. Pat. No. 5,580,737), as well as largemolecules, such as reverse transcriptase (U.S. Pat. No. 5,786,462) andthrombin (U.S. Pat. No. 5,543,293). Ribozymes are nucleic acid moleculesthat are capable of catalyzing a chemical reaction, eitherintramolecularly or intermolecularly. Ribozymes typically cleave nucleicacid substrates through recognition and binding of the target substratewith subsequent cleavage. Triplex forming function nucleic acidmolecules can interact with double-stranded or single-stranded nucleicacid by forming a triplex, in which three strands of DNA form a complexdependant on both Watson-Crick and Hoogsteen base-pairing. Triplexmolecules can bind target regions with high affinity and specificity.

The functional nucleic acid molecules may act as effectors, inhibitors,modulators, and stimulators of a specific activity possessed by a targetmolecule, or the functional nucleic acid molecules may possess a de novoactivity independent of any other molecules. In one embodiment, theantibody is a stimulator of dendritic cells

The conjugated agents can be linked to the antibody directly orindirectly, using any of a large number of available methods. Forexample, an agent can be attached at the hinge region of the reducedantibody component via disulfide bond formation, using cross-linkerssuch as N-succinyl 3-(2-pyridyldithio)proprionate (SPDP), or via acarbohydrate moiety in the Fc region of the antibody (Yu et al. 1994Int. J. Cancer 56: 244; Upeslacis et al., “Modification of Antibodies byChemical Methods,” in Monoclonal antibodies: principles andapplications, Birch et al. (eds.), pages 187-230 (Wiley-Liss, Inc.1995); Price, “Production and Characterization of SyntheticPeptide-Derived Antibodies,” in Monoclonal antibodies: Production,engineering and clinical application, Ritter et al. (eds.), pages 60-84(Cambridge University Press 1995)).

Techniques for conjugating agents to antibodies are well known (Amon etal., “Monoclonal Antibodies For Immunotargeting Of Drugs In CancerTherapy”, in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al.(eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al.,“Antibodies For Drug Delivery”, in Controlled Drug Delivery (2nd Ed.),Robinson et al, (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe,“Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review”, inMonoclonal Antibodies '84: Biological And Clinical Applications,Pinchera et al. (eds.), pp. 475-506 (1985); “Analysis, Results, AndFuture Prospective Of The Therapeutic Use Of Radiolabeted Antibody inCancer Therapy”, in Monoclonal Antibodies For Cancer Detection AndTherapy, Baldwin et al. (eds.), pp. 303-16 (Academic Press 1985), andThorpe et al., The Preparation And Cytotoxic Properties OfAntibody-Toxin Conjugates” 1982 Immunol. Rev. 62:119-58),

Antibodies or antigen-binding regions thereof can be linked to anotherfunctional molecule such as another antibody or ligand for a receptor togenerate a bi-specific or multi-specific molecule that binds to at leasttwo or more different binding sites or target molecules. Linking of theantibody to one or more other binding molecules, such as anotherantibody, antibody fragment, peptide or binding mimetic, can be done,for example, by chemical coupling, genetic fusion, or noncovalentassociation. Multi-specific molecules can further include a thirdbinding specificity, in addition to the first and second target epitope.

Bi-specific and multi-specific molecules can be prepared using methodsknown in the art. For example, each binding unit of the hi-specificmolecule can be generated separately and then conjugated to one another.When the binding molecules are proteins or peptides, a variety ofcoupling or cross-linking agents can be used for covalent conjugation.Examples of cross-linking agents include protein A, carbodiimide,N-succinimidyl-S-acetyl-thioacetate (SATA),5,5′-dithiobis(2-nitroberizoic acid) (DTNB), o-phenylenedimaleimide(oRDM), N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), andsulfosuccinimidyl 4-(N-maleimidomethyl)cyclohaxane-I-carboxylate(sulfo-SMCC) (Karpovsky et al., 1984 J. Exp. Med. 160:1686; Liu et al.,1985 Proc. Natl. Acad. Sci. USA 82:8648). When the binding molecules areantibodies, they can be conjugated by sulfhydryl bonding of theC-terminus hinge regions of the two heavy chains.

The antibodies or fragments thereof may be linked to a moiety that istoxic to a cell to which the antibody is bound to form “depleting”antibodies. These antibodies are particularly useful in applicationswhere it is desired to deplete an NK cell.

The antibodies may also be attached to solid supports, which areparticularly useful for immunoassays or purification of the targetantigen. Such solid supports include, but are not limited to, glass,cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride orpolypropylene.

The antibodies also can be bound to many different carriers. Thus,compositions are also provided containing the antibodies and anothersubstance, active or inert. Examples of well-known carriers includeglass, polystyrene, polypropylene, polyethylene, dextran, nylon,amylase, natural and modified cellulose, polyacrylamide, agarose, andmagnetite. The nature of the carrier can be either soluble or insolublefor purposes of embodiments described herein. Those skilled in the artwill know of other suitable carriers for binding monoclonal antibodies,or will be able to ascertain such, using routine experimentation.

II. CONSTRUCTS

All examples of H chain constructs are typically used in co-transfectionof CHO cells with matching L chain vectors. Also, in some embodimentsimmunotherapeutics will have humanized variable regions.

The following depicts APC-targeted antibodies and antibody-IL10 fusionproteins useful in the methods and compositions described herein.

Anti-ASGPR-49C11-hIL-10

SEQ ID NO:1 shows a fusion protein of the heavy chain of the anti-ASGPR49C11 antibody fused through a linker to human IL-10. The linker isunderlined and the IL-10 amino acid sequence is in bold italics.

mAnti-ASGPR_49C11_7H-LV-hIgG4H-C-Flex-v1-hIL-10]antibody, SEQ ID NO:1:

(SEQ ID NO: 1) VQLQESGPDLVKPSQSLSLTCTVTGYSITSGYSWHWIRQFPGNKLEWMGYILFSGSTNYNPSLKSRISITRDTSKNQFFLQLNSVTTEDTATYFCARSNYGSFASWGQGTLVTVSAAKTTGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKASQTPTNTISVTPTNNSTPTNNSNPKPNP

.

The heavy chain of the anti-ASGPR 49C11 antibody from above is SEQ IDNO:2:

(SEQ ID NO: 2) VQLQESGPDLVKPSQSLSLTCTVTGYSITSGYSWHWIRQFPGNKLEWMGYILFSGSTNYNPSLKSRISITRDTSKNQFFLQLNSVTTEDTATYFCARSNYGSFASWGQGTLVTVSAAKTTGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKAS.

The H chain variable region of anti-ASGPR 49C11 is shown in SEQ IDNO.:3:

(SEQ ID NO: 3) QLQESGPDLVKPSQSLSLTCTVTGYSITSGYSWHWIRQFPGNKLEWMGYILFSGSTNYNPSLKSRISITRDTSKNQFFLQLNSVTTEDTATYFCARSNYG SFASWGQGTLVTVSAAKTT.

The linker shown above is SEQ ID NO:4:

(SEQ ID NO: 4) QTPTNTISVTPTNNSTPTNNSNPKPNP.

The hIL-10 amino acid sequence from the Anti-ASGPR-49C11-hIL-10 is shownin SEQ ID NO:5:

(SEQ ID NO: 5) ASPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINY IEAYMTMKIRN.

The DNA sequence of the mAnti-ASGPR_49C11_7H-LV-hIgG4H-C-Flex-v1-hIL-10antibody is shown in SEQ ID NO:6:

(SEQ ID NO: 6) ATGAGAGCGCTGATTCTTTTGTGCCTGTTCACAGCCTTTCCTGGTATCCTGTCTGATGTGCAGCTTCAGGAGTCAGGACCTGACCTGGTGAAACCTTCTCAGTCACTTTCACTCACCTGCACTGTCACTGGCTACTCCATCACCAGTGGTTATAGCTGGCACTGGATCCGGCAGTTTCCAGGAAACAAACTGGAATGGATGGGCTACATACTCTTCAGTGGTAGCACTAACTACAACCCATCTCTGAAAAGTCGAATCTCTATCACTCGAGACACATCCAAGAACCAGTTCTTCCTGCAGTTGAATTCTGTGACTACTGAGGACACAGCCACATATTTCTGTGCAAGATCTAACTATGGTTCCTTTGCTTCCTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCAGCCAAAACAACGGGCCCATCCGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACGAAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGTCCAAATATGGTCCCCCATGCCCACCCTGCCCAGCACCTGAGTTCGAAGGGGGACCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAGGCTAACCGTGGACAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCTGGGTAAAGCTAGTCAGACCCCCACCAACACCATCAGCGTGACCCCCACCAACAACAGCACCCCCACCAACAACAGCAACCCCAAGCCCAACCCCGCTAGCCCAGGCCAGGGCACCCAGTCTGAGAACAGCTGCACCCACTTCCCAGGCAACCTGCCTAACATGCTTCGAGATCTCCGAGATGCCTTCAGCAGAGTGAAGACTTTCTTTCAAATGAAGGATCAGCTGGACAACTTGTTGTTAAAGGAGTCCTTGCTGGAGGACTTTAAGGGTTACCTGGGTTGCCAAGCCTTGTCTGAGATGATCCAGTTTTACCTGGAGGAGGTGATGCCCCAAGCTGAGAACCAAGACCCAGACATCAAGGCGCATGTGAACTCCCTGGGGGAGAACCTGAAGACCCTCAGGCTGAGGCTACGGCGCTGTCATCGATTTCTTCCCTGTGAAAACAAGAGCAAGGCCGTGGAGCAGGTGAAGAATGCCTTTAATAAGCTCCAAGAGAAAGGCATCTACAAAGCCATGAGTGAGTTTGACATCTTCATCAACTACATAGAAGCCTACATGACAATGAAGATA CGAAACTGA.

The corresponding light chain amino acid sequence,mAnti-ASGPR_49C11_7K-LV-hIgGK-C, is shown in SEQ ID NO:7:

(SEQ ID NO: 7) QIVLTQSPAIMSASPGEKVTMTCSASSSVSHMHWYQQKSGTSPKRWIYDTSRLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSHPWSFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVT HQGLSSPVTKSFNRGEC.

The L chain variable region of anti-ASGPR 49C11 is shown in SEQ IDNO.:8:

(SEQ ID NO: 8) QIVLTQSPAIMSASPGEKVTMTCSASSSVSHMHWYQQKSGTSPKRWIYDTSRLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSHPWSFG GGTKLE

The DNA sequence of mAnti-ASGPR_49C11_7K-LV-hIgGK-C is shown in SEQ IDNO:9:

(SEQ ID NO: 9) ATGGATTTTCAAGTGCAGATTTTCAGCTTCCTGCTAATCAGTGCCTCAGTCATAATATCCAGAGGACAAATTGTTCTCACCCAGTCTCCAGCAATCATGTCTGCATCTCCAGGGGAGAAGGTCACCATGACCTGCAGTGCCAGCTCAAGTGTAAGTCACATGCACTGGTACCAGCAGAAGTCAGGCACTTCCCCCAAAAGATGGATTTATGACACATCCAGACTGGCTTCTGGAGTCCCTGCTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTTACTCTCTCACAATCAGCAGCATGGAGGCTGAAGATGCTGCCACTTATTACTGCCAGCAGTGGAGTAGTCACCCATGGTCGTTCGGTGGAGGCACCAAACTCGAGATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTATGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAG AGTGTTAG.

Anti-CD40-24A3-hIL-10

SEQ ID NO:10 shows a fusion protein of the heavy chain of the anti-CD4024A3 antibody fused through a linker to human IL-10. The linker isunderlined and the IL-10 amino acid sequence is in bold italics.

manti-hCD40_24A3.3F1_H-LV-hIgG4H-C-Flex-v1-hIL-10 antibody, SEQ IDNO:10:

(SEQ ID NO: 10) VQLQESGPDLVKPSQSLSLTCTVTGYSITSDYSWHWIRQFPGNKLEWMGYIYYSGSTNYNPSLKSRISITRDTSKNQFFLQLNSVTTEDSATYFCARFYYGYSFFDYWGQGTTLTVSSAKTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKASQTPTNTISVTPTNNSTPTNNSNPKPNP

The heavy chain of the anti-CD40 24A3 antibody from above is SEQ IDNO:11:

(SEQ ID NO: 11) VQLQESGPDLVKPSQSLSLTCTVTGYSITSDYSWHWIRQFPGNKLEWMGYIYYSGSTNYNPSLKSRISITRDTSKNQFFLQLNSVTTEDSATYFCARFYYGYSFFDYWGQGTTLTVSSAKTKGPVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKAS.

The linker shown above is SEQ ID NO:4 and the IL-10 amino acid sequenceis shown as SEQ ID NO:5.

The DNA sequence of themanti-hCD40_24A3.3F1_H-LV-hIgG4H-C-Flex-v1-hIL-10 antibody is shown inSEQ ID NO:12:

(SEQ ID NO: 12) ATGAGAGTGCTGATTCTTTTGTGCCTGTTCACAGCCTTTCCTGGTATCCTGTCTGATGTGCAGCTTCAGGAGTCAGGACCTGACCTGGTGAAACCTTCTCAGTCACTTTCACTCACCTGCACTGTCACTGGCTACTCCATCACCAGTGATTATAGCTGGCACTGGATCCGGCAGTTCCCAGGAAACAAACTGGAATGGATGGGCTACATATATTACAGTGGTAGCACTAACTACAACCCATCTCTCAAAAGTCGAATCTCTATCACTCGAGACACATCCAAGAACCAGTTCTTCCTGCAGTTGAATTCTGTGACTACTGAGGACTCAGCCACATATTTCTGTGCAAGATTTTACTACGGTTATAGCTTCTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCAGCCAAAACAAAGGGCCCATCCGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACGAAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGTCCAAATATGGTCCCCCATGCCCACCCTGCCCAGCACCTGAGTTCGAAGGGGGACCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAGGCTAACCGTGGACAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCTGGGTAAAGCTAGTCAGACCCCCACCAACACCATCAGCGTGACCCCCACCAACAACAGCACCCCCACCAACAACAGCAACCCCAAGCCCAACCCCGCTAGCCCAGGCCAGGGCACCCAGTCTGAGAACAGCTGCACCCACTTCCCAGGCAACCTGCCTAACATGCTTCGAGATCTCCGAGATGCCTTCAGCAGAGTGAAGACTTTCTTTCAAATGAAGGATCAGCTGGACAACTTGTTGTTAAAGGAGTCCTTGCTGGAGGACTTTAAGGGTTACCTGGGTTGCCAAGCCTTGTCTGAGATGATCCAGTTTTACCTGGAGGAGGTGATGCCCCAAGCTGAGAACCAAGACCCAGACATCAAGGCGCATGTGAACTCCCTGGGGGAGAACCTGAAGACCCTCAGGCTGAGGCTACGG CGCTGTCATCGATTTCTTCCCTGTGAAAACAAGAGCAAGGCCGTGGAGCAGGTGAAGAATGCCTTTAATAAGCTCCAAGAGAAAGGCATCTACAAAGCCATGAGTGAGTTTGACATCTTCATCAACTACATAGAAGCCTACATGACAATG AAGATACGAAACTGA.

The corresponding light chain amino acid sequence,manti-hCD40_24A3.3F1_K-LV-hIgGK-C, is shown in SEQ ID NO:13:

(SEQ ID NO: 13) QIVLTOSPAFMSASPGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL SSPVTKSFNRGECAS.

The DNA sequence of manti-hCD40_24A3.3F1_K-LV-hIgGK-C is shown in SEQ IDNO:14:

(SEQ ID NO: 14) ATGGATTTTCAAGTGCAGATTTTCAGCTTCCTGCTAATCAGTGCCTCAGTCATAGTATCCAGAGGACAAATTGTTCTCACCCAGTCTCCAGCATTCATGTCTGCATCTCCAGGGGAGAAGGTCACCATGACCTGCAGTGCCAGCTCAAGTGTCAGTTACATGCACTGGTACCAGCAGAAGTCAGGCACCTCCCCCAAAAGATGGATTTATGACACATCCAAACTGGCTTCTGGAGTCCCTGCTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTTACTCTCTCACAATCAGCAGCATGGAGGCTGAAGATGCTGCCACTTATTACTGCCAGCAGTGGAGTAGTAACCCACTCACGTTCGGTGCTGGGACCAAGCTCGAGATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTATGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTGCTAGCTAG.

Anti-DCIR-9E8-hIL-10

SEQ ID NO:15 shows a fusion protein of the heavy chain of the anti-DCIR9E8 antibody fused through a linker to human IL-10. The linker isunderlined and the IL-10 amino acid sequence is in bold italics.

mAnti-DCIR_9E8_H-LV-hIgG4H-C-Flex-v1-hIL-10 antibody, SEQ ID NO:15:

(SEQ ID NO: 15) QVTLKESGPGILQPSQTLSLTCSFSGFSLSTSGMGLSWIRQPSGKGLEWLAHIYWDDDKRYNPSLKSRLTISKDTSSNQVFLKITIVDTADAATYYCARSSHYYGYGYGGYFDVWGAGTTVTVSSAKTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKASQTPTNTISVTPTNNSTPTNNSNPKPNP

The heavy chain of the anti-DCIR 9E8 antibody from above is SEQ IDNO:16:

(SEQ ID NO: 16) QVTLKESGPGILQPSQTLSLTCSFSGFSLSTSGMGLSWIRQPSGKGLEWLAHIYWDDDKRYNPSLKSRLTISKDTSSNQVFLKITIVDTADAATYYCARSSHYYGYGYGGYFDVWGAGTTVTVSSAKTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSL GKAS.

The H chain variable region of anti-DCIR 9E8 is shown in SEQ ID NO.:17:

QVTLKESGPGILQPSQTLSLTCSFSGFSLSTSGMGLSWIRQPSGKGLEWLAHIYWDDDKRYNPSLKSRLTISKDTSSNQVFLKITIVDTADAATYYCARSSHYYGYGYGGYFDVWGAGTTVTVS.

The linker shown above is SEQ ID NO:18:

(SEQ ID NO: 18) QTPTNTISVTPTNNSTPTNNSNPKPNP.

The IL-10 amino acid sequence is shown as SEQ ID NO:19:

(SEQ ID NO: 19) ASPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINY IEAYMTMKIRN.

The corresponding DNA sequence for the IL-10 gene is shown as SEQ IDNO:20:

(SEQ ID NO: 20) CGCTAGCCCAGGCCAGGGCACCCAGTCTGAGAACAGCTGCACCCACTTCCCAGGCAACCTGCCTAACATGCTTCGAGATCTCCGAGATGCCTTCAGCAGAGTGAAGACTTTCTTTCAAATGAAGGATCAGCTGGACAACTTGTTGTTAAAGGAGTCCTTGCTGGAGGACTTTAAGGGTTACCTGGGTTGCCAAGCCTTGTCTGAGATGATCCAGTTTTACCTGGAGGAGGTGATGCCCCAAGCTGAGAACCAAGACCCAGACATCAAGGCGCATGTGAACTCCCTGGGGGAGAACCTGAAGACCCTCAGGCTGAGGCTACGGCGCTGTCATCGATTTCTTCCCTGTGAAAACAAGAGCAAGGCCGTGGAGCAGGTGAAGAATGCCTTTAATAAGCTCCAAGAGAAAGGCATCTACAAAGCCATGAGTGAGTTTGACATCTTCATCAACTACATAGAAGCCTACATGACAATGAAGATACGAAACTGA.

The DNA sequence of mAnti-DCIR_9E8_H-LV-hIgG4H-C-Flex-v1-hIL-10 antibodyis shown in SEQ ID NO:21:

(SEQ ID NO: 21) ATGAACAGGCTTACTTCCTCATTGCTGCTGCTGATTGTCCCTGCATATGTCCTGTCCCAGGTTACTCTGAAAGAGTCTGGCCCTGGGATATTGCAGCCCTCCCAGACCCTCAGTCTGACTTGTTCTTTCTCTGGGTTTTCACTGAGCACTTCTGGTATGGGTCTGAGCTGGATTCGTCAGCCTTCAGGAAAGGGTCTGGAGTGGCTGGCACACATTTACTGGGATGATGACAAGCGCTATAACCCATCCCTGAAGAGCCGGCTCACAATCTCCAAGGATACCTCCAGCAACCAGGTTTTCCTCAAGATCACCATTGTGGACACTGCAGATGCTGCCACATACTACTGTGCTCGAAGCTCCCATTACTACGGTTATGGCTACGGGGGATACTTCGATGTCTGGGGCGCAGGGACCACGGTCACCGTCTCCTCAGCCAAAACGAAGGGCCCATCCGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACGAAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGTCCAAATATGGTCCCCCATGCCCACCCTGCCCAGCACCTGAGTTCGAAGGGGGACCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAGGCTAACCGTGGACAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCTGGGTAAAGCTAGTCAGACCCCCACCAACACCATCAGCGTGACCCCCACCAACAACAGCACCCCCACCAACAACAGCAACCCCAAGCCCAACCCCGCTAGCCCAGGCCAGGGCACCCAGTCTGAGAACAGCTGCACCCACTTCCCAGGCAACCTGCCTAACATGCTTCGAGATCTCCGAGATGCCTTCAGCAGAGTGAAGACTTTCTTTCAAATGAAGGATCAGCTGGACAACTTGTTGTTAAAGGAGTCCTTGCTGGAGGACTTTAAGGGTTACCTGGGTTGCCAAGCCTTGTCTGAGATGATCCAGTTTTACCTGGAGGAGGTGATGCCCCAAGCTGAGAACCAAGACCCAGACATCAAGGCGCATGTGAACTCCCTGGGGGAGAACCTGAAGACCCTCAGGCTGAGGCTACGGCGCTGTCATCGATTTCTTCCCTGTGAAAACAAGAGCAAGGCCGTGGAGCAGGTGAAGAATGCCTTTAATAAGCTCCAAGAGAAAGGCATCTACAAAGCCATGAGTGAGTTTGACATCTTCATCAACTACATAGAAGCCTACATGACAATGAAGATACGAAACTGA.

The corresponding light chain amino acid sequence,mAnti-DCIR_9E8_K-LV-hIgGK-C, is shown in SEQ ID NO:22:

(SEQ ID NO: 22) NIVLTQSPASLAVSLGQRATISCRASESIHSYGNSFLHWYQQKPGQPPKLLIYLASNLESGVPARFSGSGSRTDFTLTIDPVEADDAATYYCQQNNEDPWTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGEC.

The L chain variable region of anti-DCIR 9E8 is shown in SEQ ID NO.:23:

NIVLTQSPASLAVSLGQRATISCRASESIHSYGNSFLHWYQQKPGQPPKLLIYLASNLESGVPARFSGSGSRTDFTLTIDPVEADDAATYYCQQNNEDPW TFGGGTKLEIK.

The DNA sequence of the L chain variable region of the anti-DCIR 9E8 isshown in SEQ ID NO:24:

(SEQ ID NO: 24) AACATTGTGCTGACCCAATCTCCAGCTTCTTTGGCTGTGTCTCTAGGGCAGAGGGCCACCATATCCTGCAGAGCCAGTGAAAGTATTCATAGTTATGGCAATAGTTTTCTGCACTGGTACCAGCAGAAACCAGGACAGCCACCCAAACTCCTCATCTATCTTGCATCCAACCTAGAATCTGGGGTCCCTGCCAGGTTCAGCGGCAGTGGGTCTAGGACAGACTTCACCCTCACCATTGATCCTGTGGAGGCTGATGATGCTGCAACCTATTACTGTCAGCAAAATAATGAGGATCCGTGGACGTTCGGTGGAGGCACCAAGCTCGAGATCAAA.

The leader sequence prior to the light chain amino acid sequencecomprises: METDTLLLWVLLLWVPGSTG (SEQ ID NO:25).

The corresponding DNA sequence of the leader sequence comprises:

(SEQ ID NO: 26) ATGGAGACAGACACACTCCTGCTATGGGTGCTGCTGCTCTGGGTTCCAGGTTCCACAGGT.

The DNA sequence of mAnti-DCIR_9E8_K-LV-hIgGK-C is shown in SEQ IDNO:27:

(SEQ ID NO: 27) ATGGAGACAGACACACTCCTGCTATGGGTGCTGCTGCTCTGGGTTCCAGGTTCCACAGGTAACATTGTGCTGACCCAATCTCCAGCTTCTTTGGCTGTGTCTCTAGGGCAGAGGGCCACCATATCCTGCAGAGCCAGTGAAAGTATTCATAGTTATGGCAATAGTTTTCTGCACTGGTACCAGCAGAAACCAGGACAGCCACCCAAACTCCTCATCTATCTTGCATCCAACCTAGAATCTGGGGTCCCTGCCAGGTTCAGCGGCAGTGGGTCTAGGACAGACTTCACCCTCACCATTGATCCTGTGGAGGCTGATGATGCTGCAACCTATTACTGTCAGCAAAATAATGAGGATCCGTGGACGTTCGGTGGAGGCACCAAGCTCGAGATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTATGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCA ACAGGGGAGAGTGTTAG.

Anti-CD40-12E12-hIL-10

SEQ ID NO:28 shows a fusion protein of the heavy chain of the anti-DCIR9E8 antibody fused through a linker to human IL-10. The linker isunderlined and the IL-10 amino acid sequence is in bold italics.

mAnti-CD40_12E12.3F3_H-LV-hIgG4H-C-Flex-v1-hIL-10 antibody, SEQ IDNO:28:

(SEQ ID NO: 28) EVKLVESGGGLVQPGGSLKLSCATSGFTFSDYYMYWVRQTPEKRLEWVAYINSGGGSTYYPDTVKGRFTISRDNAKNTLYLQMSRLKSEDTAMYYCARRGLPFHAMDYWGQGTSVTVSSAKTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKASQTPTNTISVTPTNNSTPTNNSNPKPNP

.

The heavy chain of the anti-CD40 12E12 antibody from above is SEQ IDNO:29:

(SEQ ID NO: 29) EVKLVESGGGLVQPGGSLKLSCATSGFTFSDYYMYWVRQTPEKRLEWVAYINSGGGSTYYPDTVKGRFTISRDNAKNTLYLQMSRLKSEDTAMYYCARRGLPFHAMDYWGQGTSVTVSSAKTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKAS.

The H chain variable region of anti-CD40 12E12 is shown in SEQ IDNO.:30:

EVKLVESGGGLVQPGGSLKLSCATSGFTFSDYYMYWVRQTPEKRLEWVAYINSGGGSTYYPDTVKGRFTISRDNAKNTLYLQMSRLKSEDTAMYYCARRG LPFHAMDYWGQGTSVTVS.

The CDRs of the The H chain variable of anti-CD40 12E12 are:

(SEQ ID NO: 31) CDR1: SASQGISNYLN, (SEQ ID NO: 32)CDR2: AYINSGGGSTYYPDTVK, and (SEQ ID NO: 33) CDR3: RRGLPFHAMD.

The linker shown above is SEQ ID NO:4 and the IL-10 amino acid sequenceis shown as SEQ ID NO:5.

The DNA sequence of mAnti-CD40_12E12.3F3_H-LV-hIgG4H-C-Flex-v1-hIL-10antibody is shown in SEQ ID NO:34:

(SEQ ID NO: 34) ATGAACTTGGGGCTCAGCTTGATTTTCCTTGTCCTTGTTTTAAAAGGTGTCCAGTGTGAAGTGAAGCTGGTGGAGTCTGGGGGAGGCTTAGTGCAGCCCGGAGGGTCCCTGAAACTCTCCTGTGCAACCTCTGGATTCACTTTCAGTGACTATTACATGTATTGGGTTCGCCAGACTCCAGAGAAGAGGCTGGAGTGGGTCGCATACATTAATTCTGGTGGTGGTAGCACCTATTATCCAGACACTGTAAAGGGCCGATTCACCATCTCCAGAGACAATGCCAAGAACACCCTGTACCTGCAAATGAGCCGGCTGAAGTCTGAGGACACAGCCATGTATTACTGTGCAAGACGGGGGTTACCGTTCCATGCTATGGACTATTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCAGCCAAAACGAAGGGCCCATCCGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACGAAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGTCCAAATATGGTCCCCCATGCCCACCCTGCCCAGCACCTGAGTTCGAAGGGGGACCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAGGCTAACCGTGGACAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCTGGGTAAAGCTAGTCAGACCCCCACCAACACCATCAGCGTGACCCCCACCAACAACAGCACCCCCACCAACAACAGCAACCCCAAGCCCAACCCCGCTAGCCCAGGCCAGGGCACCCAGTCTGAGAACAGCTGCACCCACTTCCCAGGCAACCTGCCTAACATGCTTCGAGATCTCCGAGATGCCTTCAGCAGAGTGAAGACTTTCTTTCAAATGAAGGATCAGCTGGACAACTTGTTGTTAAAGGAGTCCTTGCTGGAGGACTTTAAGGGTTACCTGGGTTGCCAAGCCTTGTCTGAGATGATCCAGTTTTACCTGGAGGAGGTGATGCCCCAAGCTGAGAACCAAGACCCAGACATCAAGGCGCATGTGAACTCCCTGGGGGAGAACCTGAAGACCCTCAGGCTGAGGCTACGGCGCTGTCATCGATTTCTTCCCTGTGAAAACAAGAGCAAGGCCGTGGAGCAGGTGAAGAATGCCTTTAATAAGCTCCAAGAGAAAGGCATCTACAAAGCCATGAGTGAGTTTGACATCTTCATCAACTACATAGAAGCCTACATGACA ATGAAGATACGAAACTGA.

The corresponding light chain amino acid sequence,mAnti-CD40_12E12.3F3_K-V-hIgGK-C, is shown in SEQ ID NO:35:

(SEQ ID NO: 35) DIQMTQTTSSLSASLGDRVTISCSASQGISNYLNWYQQKPDGTVKLLIYYTSILHSGVPSRFSGSGSGTDYSLTIGNLEPEDIATYYCQQFNKLPPTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC.

The L chain variable region of anti-CD40 12E12 is shown in SEQ IDNO.:36:

(SEQ ID NO: 36) DIQMTQTTSSLSASLGDRVTISCSASQGISNYLNWYQQKPDGTVKLLIYYTSILHSGVPSRFSGSGSGTDYSLTIGNLEPEDIATYYCQQFNKLPPTFGG GTKLEIK.

The CDRs of the L chain variable of anti-CD40 12E12 are CDR1:SASQGISNYLN (SEQ ID NO:37), CDR2: YTSILHS (SEQ ID NO:38), and CDR3:QQFNKLPPT (SEQ ID NO:39).

The DNA sequence of mAnti-CD40_12E12.3F3_K-V-hIgGK-C is shown in SEQ IDNO:40:

(SEQ ID NO: 40) ATGATGTCCTCTGCTCAGTTCCTTGGTCTCCTGTTGCTCTGTTTTCAAGGTACCAGATGTGATATCCAGATGACACAGACTACATCCTCCCTGTCTGCCTCTCTAGGAGACAGAGTCACCATCAGTTGCAGTGCAAGTCAGGGCATTAGCAATTATTTAAACTGGTATCAGCAGAAACCAGATGGAACTGTTAAACTCCTGATCTATTACACATCAATTTTACACTCAGGAGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGGACAGATTATTCTCTCACCATCGGCAACCTGGAACCTGAAGATATTGCCACTTACTATTGTCAGCAGTTTAATAAGCTTCCTCCGACGTTCGGTGGAGGCACCAAACTCGAGATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTATGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG.

IgG-hIL10 Control

SEQ ID NO:41 shows a fusion protein of the heavy chain of the IgGcontrol antibody fused through a linker to human IL-10. The linker isunderlined and the IL-10 amino acid sequence is in bold italics.

hIgG4H-Flex-v1-hIL-10 antibody, SEQ ID NO:41:

(SEQ ID NO: 41) RLQLQESGPGLLKPSVTLSLTCTVSGDSVASSSYYWGWVRQPPGKGLEWIGTINFSGNMYYSPSLRSRVTMSADMSENSFYLKLDSVTAADTAVYYCAAGHLVMGFGAHWGQGKLVSVSPASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKASQTPTNTISVTPTNNSTPTNNSNPKPNP

.

The linker shown above is SEQ ID NO:4 and the IL-10 amino acid sequenceis shown as SEQ ID NO:5.

The DNA sequence of hIgG4H-Flex-v1-hIL-10 antibody antibody is shown inSEQ ID NO:42:

(SEQ ID NO: 42) ATGGACCTCCTGTGCAAGAACATGAAGCACCTGTGGTTCTTCCTCCTGCTGGTGGCGGCTCCCAGATGGGTCCTGTCCCGGCTGCAGCTGCAGGAGTCGGGCCCAGGCCTGCTGAAGCCTTCGGTGACCCTGTCCCTCACCTGCACTGTCTCGGGTGACTCCGTCGCCAGTAGTTCTTATTACTGGGGCTGGGTCCGTCAGCCCCCAGGGAAGGGACTCGAGTGGATAGGGACTATCAATTTTAGTGGCAATATGTATTATAGTCCGTCCCTCAGGAGTCGAGTGACCATGTCGGCAGACATGTCCGAGAACTCCTTCTATCTGAAATTGGACTCTGTGACCGCAGCAGACACGGCCGTCTATTATTGTGCGGCAGGACACCTCGTTATGGGATTTGGGGCCCACTGGGGACAGGGAAAACTGGTCTCCGTCTCTCCAGCTTCCACCAAGGGCCCATCCGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACGAAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGTCCAAATATGGTCCCCCATGCCCACCCTGCCCAGCACCTGAGTTCGAAGGGGGACCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAGGCTAACCGTGGACAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCTGGGTAAAGCTAGTCAGACCCCCACCAACACCATCAGCGTGACCCCCACCAACAACAGCACCCCCACCAACAACAGCAACCCCAAGCCCAACCCCGCTAGCCCAGGCCAGGGCACCCAGTCTGAGAACAGCTGCACCCACTTCCCAGGCAACCTGCCTAACATGCTTCGAGATCTCCGAGATGCCTTCAGCAGAGTGAAGACTTTCTTTCAAATGAAGGATCAGCTGGACAACTTGTTGTTAAAGGAGTCCTTGCTGGAGGACTTTAAGGGTTACCTGGGTTGCCAAGCCTTGTCTGAGATGATCCAGTTTTACCTGGAGGAGGTGATGCCCCAAGCTGAGAACCAAGACCCAGACATCAAGGCGCATGTGAACTCCCTGGGGGAGAACCTGAAGACCCTCAGGCTGAGGCTACGGCGCTGTCATCGATTTCTTCCCTGTGAAAACAAGAGCAAGGCCGTGGAGCAGGTGAAGAATGCCTTTAATAAGCTCCAAGAGAAAGGCATCTACAAAGCCATGAGTGAGTTTGACATCTTCATCAACTACATAGAAGCCTACATGACAATGAAGATACGAAACTGA.

The corresponding light chain amino acid sequence hIgGK, is shown in SEQID NO:43:

(SEQ ID NO: 43) DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY ACEVTHQGLSSPVTKSFNRGEC.

The DNA sequence of hIgGK is shown in SEQ ID NO:44:

(SEQ ID NO: 44) ATGAGGGTCCCCGCTCAGCTCCTGGGGCTCCTGCTACTCTGGCTCCGAGGTGCCAGATGTGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTCCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAGTTACAGTACCCCGTACACTTTTGGCCAGGGGACCAAGCTGGAGATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG.

Shown below are further examples of antibodies and antibody fragmentsuseful in the methods and compositions described herein.

Anti-Dectin-1 mAbs

manti-Dectin-1-11B6.4-H-V-hIgG4H-C]; SEQ ID NO:45:

(SEQ ID NO: 45) QVQLKESGPGLVAPSQSLSITCSVSGFSLSNYDISWIRQPPGKGLEWLGVMWTGGGANYNSAFMSRLSINKDNSKSQVFLKMNNLQTDDTAIYYCVRDAVRYWNFDVWGAGTTVTVSSAKTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHN HYTQKSLSLSLGKAS.

The above sequence is a chimera between the H chain variable region ofthe mAb 11B6.4 and the C region of hIgG4.

The H chain variable region of the mAb 11B6.4 is shown in SEQ ID NO:46:

(SEQ ID NO: 46) QVQLKESGPGLVAPSQSLSITCSVSGFSLSNYDISWIRQPPGKGLEWLGVMWTGGGANYNSAFMSRLSINKDNSKSQVFLKMNNLQTDDTAIYYCVRDAVRYWNFDVWGAGTTVTVSSAKTK.

[manti-Dectin-1-11B6.4-K-LV-hIgGK-C] is the corresponding L chainchimera; SEQ ID NO:47:

(SEQ ID NO: 47) QIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWYQQKPGSSPKPWIYATSHLASGVPARFSGSGSGTSYSLTISRVEAEDTATYYCQQWSSNPFTFGSGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYA CEVTHQGLSSPVTKSFNRGEC.

The L chain variable region of the manti-Dectin-1-11B6.4-K-LV-hIgGK-C isshown in SEQ ID NO:48:

(SEQ ID NO: 48) QIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWYQQKPGSSPKPWIYATSHLASGVPARFSGSGSGTSYSLTISRVEAEDTATYYCQQWSSNPFT FGSGTK.

manti-Dectin-1-15E2.5-H-V-hIgG4H-C]; SEQ ID NO:49:

(SEQ ID NO: 49) QVQLQQSGAELARPGASVKMSCKASGYTFTTYTMHWVKQRPGQGLEWIGYINPSSGYTNYNQKFKDKATLTADKSSSTASMQLSSLTSEDSAVYYCARERAVLVPYAMDYWGQGTSVTVSSAKTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEA LHNHYTQKSLSLSLGKAS.

The above sequence is a chimera between the H chain variable region ofthe mAb 15E2.5 and the C region of hIgG4.

The H chain variable region of the mAb 15E2.5 is shown in SEQ ID NO:50:

(SEQ ID NO: 50) QVQLQQSGAELARPGASVKMSCKASGYTFTTYTMHWVKQRPGQGLEWIGYINPSSGYTNYNQKFKDKATLTADKSSSTASMQLSSLTSEDSAVYYCARERAVLVPYAMDYWGQGTSVTVSSAKTK.

[manti-Dectin-1-15E2.5-K-V-hIgGK-C] is the corresponding L chainchimera; SEQ ID NO:51:

(SEQ ID NO: 51) QIVLTQSPAVMSASPGEKVTITCTASSSLSYMHWFQQKPGTSPKLWLYSTSILASGVPTRFSGSGSGTSYSLTISRMEAEDAATYYCQQRSSSPFTFGSGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYA CEVTHQGLSSPVTKSFNRGEC.

The L chain variable region of the manti-Dectin-1-15E2.5-K-V-hIgGK-C isshown in SEQ ID NO:52:

(SEQ ID NO: 52) QIVLTQSPAVMSASPGEKVTITCTASSSLSYMHWFQQKPGTSPKLWLYSTSILASGVPTRFSGSGSGTSYSLTISRMEAEDAATYYCQQRSSSPFT FGSGTK.

manti-Dectin-1-2D8.2D4-H-V-hIgG4H-C]; SEQ ID NO:53:

(SEQ ID NO: 53) EVQLQQSGPELEKPGASVKISCKASGYSFTGYNMNWVKQSNGKSLEWIGNIDPYYGDTNYNQKFKGKATLTVDKSSSTAYMHLKSLTSEDSAVYYCARPYGSEAYFAYWGQGTLVTVSAAKTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALH NHYTQKSLSLSLGKAS.

The above sequence is a chimera between the H chain variable region ofthe mAb 2D8.2D4 and the C region of hIgG4.

The H chain variable region of the mAb 2D8.2D4 is shown in SEQ ID NO:54:

(SEQ ID NO: 54) EVQLQQSGPELEKPGASVKISCKASGYSFTGYNMNWVKQSNGKSLEWIGNIDPYYGDTNYNQKFKGKATLTVDKSSSTAYMHLKSLTSEDSAVYYCARPYGSEAYFAYWGQGTLVTVSAAKTK.

[manti-Dectin-1-2D8.2D4-K-V-hIgGK-C] is the corresponding L chainchimera; SEQ ID NO:55:

(SEQ ID NO: 55) DIVMTQSPATLSVTPGDRVSLSCRASQSISDYLHWYQQKSHESPRLLIKYAAQSISGIPSRFSGSGSGSDFTLSINGVEPEDVGVYYCQNGHSFPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGEC.

The L chain variable region of the manti-Dectin-1-2D8.2D4-K-V-hIgGK-C isshown in SEQ ID NO:56:

(SEQ ID NO: 56) DIVMTQSPATLSVTPGDRVSLSCRASQSISDYLHWYQQKSHESPRLLIKYAAQSISGIPSRFSGSGSGSDFTLSINGVEPEDVGVYYCQNGHSFPYTF GGGTK.

Anti-DC ASGPR mAbs

[mAnti-ASGPR-4G2.2-Hv-V-hIgG4H-C]; SEQ ID NO.:57:

(SEQ ID NO: 57) QIQLVQSGPELKKPGETVKISCKASGYTFTNYGMNWVKQVPGKGLRWMGWMDTFTGEPTYADDFKGRFAFSLETSASTAYLQINSLKNEDTATYFCARGGILRLNYFDYWGQGTTLTVSSAKTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSL SLSLGKAS.

The above sequence is a chimera between the H chain variable of the mAb4G2.2 and the C region of hIgG4.

The H chain variable of the mAb 4G2.2 is shown in SEQ ID NO.:58:

(SEQ ID NO: 58) QIQLVQSGPELKKPGETVKISCKASGYTFTNYGMNWVKQVPGKGLRWMGWMDTFTGEPTYADDFKGRFAFSLETSASTAYLQINSLKNEDTATYFCARGGILRLNYFDYWGQGTTLTVSSAKTK.

[mAnti-ASGPR-4G2.2-Kv-V-hIgGK-C] is the corresponding L chain chimera;SEQ ID NO.:59:

(SEQ ID NO: 59) DIQMTQSSSSFSVSLGDRVTITCKASEDIYNRLGWYQQKPGNAPRLLISGATSLETGVPSRFSGSGSGKDYALSITSLQTEDLATYYCQQCWTSPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGEC.

The L chain variable region of the mAnti-ASGPR-4G2.2-Kv-V-hIgGK-C isshown in SEQ ID NO.:60:

(SEQ ID NO: 60) DIQMTQSSSSFSVSLGDRVTITCKASEDIYNRLGWYQQKPGNAPRLLISGATSLETGVPSRFSGSGSGKDYALSITSLQTEDLATYYCQQCWTSPYTF GGGTKLEI.

[mAnti-ASGPR-5F10H-LV-hIgG4H-C] (SEQ ID NO.:61):

(SEQ ID NO: 61) EVQLQQSGPELVKPGASVKMSCKASGYTFTDYYMKWVKQSHGKSLEWIGDINPNYGDTFYNQKFEGKATLTVDKSSRTAYMQLNSLTSEDSAVYYCGRGDYGYFDVWGAGTTVTVSSAKTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLLSL GKAS.

The above sequence is a chimera between the H chain variable of the mAb5F10 and the C region of hIgG4.

The H chain variable of the mAb 5F10 is shown in SEQ ID NO.:62:

(SEQ ID NO: 62) EVQLQQSGPELVKPGASVKMSCKASGYTFTDYYMKWVKQSHGKSLEWIGDINPNYGDTFYNQKFEGKATLTVDKSSRTAYMQLNSLTSEDSAVYYCGRGDYGYFDVWGAGTTVTVSSAKTK.

[mAnti-ASGPR-5F10K-LV-hIgGK-C] is the corresponding L chain chimera; SEQID NO.:63:

(SEQ ID NO: 63) DIVMTQSHKFMSTSVGDRVSITCKASQDVGTAVAWYQQKPGQSPKLLIYWASTRHTGVPDRFTGSGSGTDFTLTINNVOSEDLADYFCQQYSSNPYMFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGEC.

The L chain variable region of the mAnti-ASGPR-5F10K-LV-hIgGK-C is shownin SEQ ID NO.:64:

(SEQ ID NO: 64) DIVMTQSHKFMSTSVGDRVSITCKASQDVGTAVAWYQQKPGQSPKLLIYWASTRHTGVPDRFTGSGSGTDFTLTINNVQSEDLADYFCQQYSSNPYMF GGGTKLEI.

[mAnti-ASGPR-1H11H-V-hIgG4H-C] (SEQ ID NO.:65):

(SEQ ID NO: 65) QLQQSGPELVKPGASVKISCKTSGYTFTEYTMHWVRQSHGKSLEWIGGINPINGGPTYNQKFKGKATLTVDKSSSTAYMELRSLTSEDSAVYYCARWDYGSRDVMDYWGQGTSVTVSSAKTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSL SLGKAS.

The above sequence is a chimera between the H chain variable of the mAb1H11 and the C region of hIgG4.

The H chain variable of the mAb 1H11 is shown in SEQ ID NO.:66:

(SEQ ID NO: 66) QLQQSGPELVKPGASVKISCKTSGYTFTEYTMHWVRQSHGKSLEWIGGINPINGGPTYNQKFKGKATLTVDKSSSTAYMELRSLTSEDSAVYYCARWDYGSRDVMDYWGQGTSVTVSSAKTK.

[mAnti-ASGPR-1H11K-LV-hIgGK-C] is the corresponding L chain chimera, SEQID NO.:67:

(SEQ ID NO: 67) NIVMTQSPKSMSMSVGERVTLSCKASENVGTYVSWYQQRPEQSPKLLIYGASNRYTGVPDRFTGSGSATDFTLTISSVQAEDLADYHCGQTYSYIFTFGSGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGEC.

The L chain variable region of the mAnti-ASGPR-1H11K-LV-hIgGK-C is shownin SEQ ID NO.:68:

(SEQ ID NO: 68) NIVMTQSPKSMSMSVGERVTLSCKASENVGTYVSWYQQRPEQSPKLLIYGASNRYTGVPDRFTGSGSATDFTLTISSVQAEDLADYHCGQTYSYIFTF GSGTKLE.

manti-hASGPR-6.3H9.1D11H (heavy chain) SEQ ID NO: 69:

(SEQ ID NO: 69) VQLQQSGAELVRPGTSVKMSCEAARFTFSNYWIGWVKQRPGHGLEWIGDIFPGGDYTNYNKKFKDKATLTADTSSSTAYMQLSSLTSEDSAIYYCARSDYGGYYVFDYWGQGTTLTVSSAKTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLS LSLGK.

manti-hASGPR_6.3H9.1D11K (light chain) SEQ ID NO: 70:

(SEQ ID NO: 70) DIVMSQSPSSLAVSVGEKVTMSCKSSQNLLYSSNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYSYPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

manti-hASGPR-5H8.1D4H (heavy chain) SEQ ID NO: 71:

(SEQ ID NO: 71) AQIQLVQSGPELKKPGETVKISCKASGYTFTDYSVHWVKQAPGKGLKWMGWINTETGEPTYADDLKGRFAFSLETSASTAYLQINNLKNEDTATYFCAKPTYRFFDYWGQGTTLTASSAKTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSL SLGK.

manti-hASGPR-5H8.1D4K (light chain) SEQ ID NO: 72:

(SEQ ID NO: 72) DIVMSQSPSSLAVSAGEKVTMSCKSSQSLLNSRTRKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCKQSYNLWTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

anti-CD40 mAbs

anti-CD40-12B4.2C10, heavy chain, (SEQ ID NO:73:

(SEQ ID NO: 73) MEWSWIFLFLLSGTAGVHSEVQLQQSGPELVKPGASVKMSCKASGYTFTDYVLHWVKQKPGQGLEWIGYINPYNDGTKYNEKFKGKATLTSDKSSSTAYMELSSLTSEDSAVYYCARGYPAYSGYAMDYWGQGTSVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAV LQKGEFV.

anti-CD40-12B4.2C10, light chain, SEQ ID NO:74:

(SEQ ID NO: 74) MMSSAQFLGLLLLCFQGTRCDIQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCHHGNTLPWTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC.

anti-CD40-12B4.2C10, light chain—alternative clone (17K6), SEQ ID NO:75:

(SEQ ID NO: 75) MDFQVQIFSFLLISASVIIVISRGQIVLTQSPAILSASPGEKVTMTCSASSSVSYMYRYQQKPGSSPKPWIYGTSNLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQYHSYPLTFGAGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC.

anti-CD40_11B6.1C3, heavy chain, SEQ ID NO:76:

(SEQ ID NO: 76) MGWSWIFLFLLSGTAGVLSEVQLQQSGPELVKPGASVKISCKASGYSFTGYYMHWVKQSHVKSLEWIGRINPYNGATSYNQNFKDKASLTVDKSSSTAYMELHSLTSEDSAVYYCAREDYVYWGQGTTLTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQKGEFV.

anti-CD40_11B6.1C3, light chain, SEQ ID NO:77:

(SEQ ID NO: 77) MKLPVRLLVLMFWIPASSSDVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFALKISRVEAEDLGVYFCSQSTHVPWTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC.

anti-LOX-1 Abs

[mAnti-LOX-1-11C8H-LV-hIgG4H-C], heavy chain, SEQ ID NO:78:

(SEQ ID NO: 78) EVQLQQSGTVLARPGASVKMSCKASGYTFTSYWMHWVKQRPGQGLEWIGAIYPGNSDTTYNQKFKGKAKLTAVTSTSTAYMELSSLTNEDSAVYYCTPTYYFDYWGQGTSLTVSSAKTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG KAS.

The H chain variable of the Ab 11C8 is shown in SEQ ID NO:79:

(SEQ ID NO: 79) EVQLQQSGTVLARPGASVKMSCKASGYTFTSYWMHWVKQRPGQGLEWIGAIYPGNSDTTYNQKFKGKAKLTAVTSTSTAYMELSSLTNEDSAVYYCTPTYYFDYWGQGTSLTVSSAKTK.

[mAnti-LOX-1-11C8K-LV-hIgGK-C], light chain, SEQ ID NO:80:

(SEQ ID NO: 80) DVVMTQTPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWFLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPWTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

The L chain variable of the Ab 11C8 is shown in SEQ ID NO:81:

(SEQ ID NO: 81) DVVMTQTPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWFLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCW QGTHFPWTFGGGTKLE

[mAnti-LOX-1-10F9H-LV-hIgG4H-C], heavy chain, SEQ ID NO:82:

(SEQ ID NO: 82) QVQLQQSGAELMKPGASVKISCKATGYTFGSYWIEWVKQRPGHGLEWIGEILPGSGNTNYNENFKGKATFTADTSSNTAYMQLTSLTSEDSAVYYCARAGIYWGQGTLVTVSAAKTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHE ALHNHYTQKSLSLSLGKAS.

The H chain variable of the Ab 10F9 is shown in SEQ ID NO:83:

(SEQ ID NO: 83) QVQLQQSGAELMKPGASVKISCKATGYTFGSYWIEWVKQRPGHGLEWIGEILPGSGNTNYNENFKGKATFTADTSSNTAYMQLTSLTSEDSAVYYCARAGIYWGQGTLVTVSAAKTK.

[mAnti-LOX-1-10F9K-LV-hIgGK-C], light chain, SEQ ID NO:84:

(SEQ ID NO: 84) DIVLTQSPAFLAVSLGQRATISCRASESVDNYGISFMNWFQQKPGQPPKLLIYVASKQGSGVPARFSGSGSGTDFSLNIHPMEEDDTAMYFCQQSKEVPRTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

The L chain variable of the Ab 10F9 is shown in SEQ ID NO:85:

(SEQ ID NO: 85) DIVLTQSPAFLAVSLGQRATISCRASESVDNYGISFMNWFQQKPGQPPKLLIYVASKQGSGVPARFSGSGSGTDFSLNIHPMEEDDTAMYFCQQSKEVPR TFGGGTKLE.

[mAnti-LOX-1-15C4H-LV-hIgG4H-C], heavy chain, SEQ ID NO:86:

(SEQ ID NO: 86) EIQLQQTGPELVKPGASVKISCKASGYPFTDYEVIVWVKQSHGKSLEWIGNISPYYGTTNYNLKFKGKATLTVDKSSSTAYMQLNSLTSEDSAVYYCARSPNWDGAWFAHWGQGALVTVSAAKTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKAS.

The H chain variable of the Ab 15C4 is shown in SEQ ID NO:87:

(SEQ ID NO: 87) EIQLQQTGPELVKPGASVKISCKASGYPFTDYIMVWVKQSHGKSLEWIGNISPYYGTTNYNLKFKGKATLTVDKSSSTAYMQLNSLTSEDSAVYYCARSPNWDGAWFAHWGQGALVTVSAAKTK.

[mAnti-LOX-1-15C4K-LV-hIgGK-C], light chain, SEQ ID NO: 88:

(SEQ ID NO: 88) DIVLTQSPASLAVSLGQRATISCKASQSVDYDGDSYMNWFQQKPGQPPKLLIYAASNLESGIPARFSGSGSGTDFTLNIHPVEEEDAATYYCQQSNEDPFTFGSGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGEC.

The L chain variable of the Ab 15C4 is shown in SEQ ID NO:89:

(SEQ ID NO: 89) DIVLTQSPASLAVSLGQRATISCKASQSVDYDGDSYMNWFQQKPGQPPKLLIYAASNLESGIPARFSGSGSGTDFTLNIHPVEEEDAATYYCQQSNEDPF TFGSGTKLE.

Anti-DCIR Abs

Anti-DCIR_24A5.4A5_H-V-hIgG4H-C, heavy chain, SEQ ID NO:90:

(SEQ ID NO: 90) MDWLWNLLFLMAAAQSAQAQIQLVQSGPELKKPGETVKISCKASGYSFTNYGMNWVKQAPGKGLKWMGWINTYTGESTYADDFKGRFAFSLETSASTAYLQISNLKNEDMATYFCARGDFRYYYFDYWGQGTTLTGSSAKTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKAS.

Anti-DCIR_24A5.4A5_K-V-hIgGK-C, light chain, SEQ ID NO:91:

(SEQ ID NO: 91) MSVLTQVLALLLLWLTGARCDIQMTQSPASLSASVGETVTITCRASGNIHNYLAWYQQKQGKSPQLLVYNAKTLADGVPSRFSGSGSGTQYSLKINTLQPEDFGSYYCQHFWDSWTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

Anti-DCIR_24E7.3H9_H-V-hIgG4H-C, heavy chain, SEQ ID NO:92:

(SEQ ID NO: 92) MEWTWVFLFLLSVTAGVHSQVQLQQSGAELMKPGASVKISCKATGYTFSSYWIEWVKQRPGHGLEWIGEILPGSGRTNDNEKFKGKATFTADTSSKKAYMQLSSLTSEDSAVYYCARRGGYSFAYWGQGTLVTVSAAKTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKAS.

Anti-DCIR_24E7.3H9_K-V-hIgGK-C, light chain, SEQ ID NO:93:

(SEQ ID NO: 93) MTMFSLALLLSLLLLCVSDSRAETTVTQSPASLSMAIGEKVTIRCVTSTDIDDDVNWYQQKPGEPPKLLISEGNTLRPGVPSRFSSSGYGTDFVFTIENMLSEDVADYYCLQSGNLPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

Anti-DCIR_29E9.2E2_H-VhIgG4H-C, heavy chain, SEQ ID NO:94:

(SEQ ID NO: 94) MAWVWTLLFLMAAAQSAQAQIQLVQSGPELKKPGETVKISCKASGYTFTNYGMNWVKQAPGKGLKWVGWINTFTGEPTYVDDFKGRFAFSLETSASTAYLQINNLKNEDTATYFCARGNFRYYYFDYWGQGTTLTVSSAKTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEAL HNHYTQKSLSLSLGKAS.

Anti-DCIR_29E9.2E2_K-V-hIgGK-C, light chain, SEQ ID NO:95:

(SEQ ID NO: 95) MSVLTQVLALLLLWLTGARCDIQMTQSPASLSASVGETVTITCRTSGNIRNYLAWYQQKQGKSPQLLVYNAKTLADGVPSRFGGSGSGTQYSLKINSLQPEDFGNYYCQHFWSSPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

Anti-DCIR_29 G10.3D9_H-V-hIgG4H-C, heavy chain, SEQ ID NO:96:

(SEQ ID NO: 96) MMGWSYIILFLVATATDVHSQVQLQQPGAELVKPGASVKLSCKASGYTFTSYWMHWVKQRPGEGLEWIGEINPSYGRTDYNEKFKNKATLTVAKSSSTAYMQLSSLTSEDSAVYYCARGDYYGSSSFAYWGQGTLVTVSAAKTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKAS 

Anti-DCIR_29 G10.3D9_K-Var1-V-hIgGK-C, light chain, SEQ ID NO:97:

(SEQ ID NO: 97) MDFQVQIFSFLLMSASVEVISRGQIVLTQSPALMSASPGEKVTMTCSASSNISYMYWYQQKPRSSPKPWIYLTSNLASGVPARFSGSGSGTSYSLTTSSMEAEDAATYCCQQWSSNPPTFGAGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

Anti-DCIR_29 G10.3D9_K-Var2-V-hIgGK-C, light chain, SEQ ID NO:98:

(SEQ ID NO: 98) MDFRVQIFSFLLMSASVEVISRGQIVLTQSPALMSASPGEKVTMTCSASSNISYMYWYQQKPRSSPKPWIYLTSNLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPPTFGAGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

Anti-DCIR_31A6.1F5_H-var2-V-hIgG4H-C, heavy chain, SEQ ID NO:99:

(SEQ ID NO: 99) MECNWILPFILSVISGVYSEVQLQQSGTVLARPGASVNMSCKAAGYSFTSYWVYWVKQRPGQGLEWIGAIYPKNSRTSYNQKFQDKATLTAVTSASTAYMELSSLTNEDSAVYYCTRPHYDSFGYWGQGTLVTVSAAKTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHN HYTQKSLSLSLGKAS.

Anti-DCIR_31A6.1F5_K-var2-V-hIgGK-C, light chain, SEQ ID NO:100:

(SEQ ID NO: 100) METDTLLLWVLLLWVPGSTGDIVLTQSPASLAVSLGQRATISCRASESVDSYGISFMHWYQQKPGQPPKLLIYRASNQESGIPARFSGSGSRTDFTLTINPVEADDVATYYCQQSNEDPLTFGAGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

Anti-DCIR_3C2.2D9_H-LV-hIgG4H-C, heavy chain, SEQ ID NO:101:

(SEQ ID NO: 101) NRLTSSLLLLIVPAYVLSQQVTLKESGPGILQPSQTLSLTCSFSGFSLSTSGMGVSWIRQPSGKGLEWLAHIYWDDDKRYNPSLKSRLTIFKDPSSNQVFLRITSVDTADTATYYCARNSHYYGSTYGGYFDVWGAGTTVTVSSAKTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKAS.

Anti-DCIR_3C2.2D9_K-LV-hIgGK-C, light chain, SEQ ID NO:102:

(SEQ ID NO: 102) METDTLLLWVLLLGVPGSTGNIVLTQSPTSFTVSLGQRATISCRASESVHSYGNSFMHWYQQKPGQPPKLLIYLASNVESGVPARFSGSGSRTDFTLTIDPVEADDAATYYCQQNSEDPWTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

Anti-DCIR_6C8.1G9_H-V-hIgG4H-C, heavy chain, SEQ ID NO:103:

(SEQ ID NO: 103) MEWTWVFLFLLSVTAGVHSQVQLQQSGTELMKPGASVKISCKATGYTFSTYWIEWVKQRPGHGLEWIGEILPGSGRTNDNEKFKGKATITADTSSKKAYMQLSSLTSEDSAVYYCARRGGYSFAFWGQGTLVSVSAAKTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKAS.

Anti-DCIR_6C8.1G9_K-V-hIgGK-C, light chain, SEQ ID NO:104:

(SEQ ID NO: 104) MTMFSLALLLSLLLLCVSDSRAETTVTQSPASLSMAIGEKVTIRCVTSTDIDDDVNWYQQKPGEPPKLLISEGNTLRAGVPSRFSSSGYGTDFVFTIENMLSEDVADYYCLQSGNLPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

Anti-DCIR2C9H-LV-hIgG4H-V-hIgG4H-C, heavy chain, SEQ ID NO:105:

(SEQ ID NO: 105) MKCSWVIFFLMAVVTGVNSEVQLQQSGAELVRPGALVKLSCKASGFNINDYYIHWVKQRPEQGLERIGWIDPDNGNTIYDPKFQGKASITADTSPNTAYLQLSSLTSEDTAVYYCARTRSPMVTTGFVYWGQGTVVTVSAAKTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKXKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK.

Anti-DCIR_2C9K-V-hIgGK-C, light chain, SEQ ID NO:106:

(SEQ ID NO: 106) METDTLLLWVLLLWVPGSTGDIVLIQSPASLAVSLGQRATISCRASESVDSYVNSFMHWYQQKPGQPPKLLIYRVSNLESGIPARFSGSGSRTDFTLTINPVEADDVATYYCQQSNEDPFTFGSGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

Anti-Langerin Abs

Anti-Langerin-15B10H-LV-hIgG4H-C, heavy chain, SEQ ID NO:107:

(SEQ ID NO: 107) QVQLRQSGPELVKPGASVKMSCKASGYTFTDYVISWVKQRTGQGLEWIGDIYPGSGYSFYNENFKGKATLTADKSSTTAYMQLSSLTSEDSAVYFCATYYNYPFAYWGQGTLVTVSAAKTTGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLS LGKAS.

The H chain variable of the Ab 15B10 is shown in SEQ ID NO:108:

(SEQ ID NO: 108) SVKMSCKASGYTFTDYVISWVKQRTGQGLEWIGDIYPGSGYSFYNENFKGKATLTADKSSTTAYMQLSSLTSEDSAVYFCA.

Anti-Langerin-15B10K-LV-hIgGK-C, light chain, SEQ ID NO:109:

(SEQ ID NO: 109) DVVMTQTPLSLPVRLGDQASISCRSSQSLVHSNGNTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTNFTLKISRVEAEDLGLYFCSQSTHVPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

The L chain variable of the Ab 15B10 is shown in SEQ ID NO:110:

(SEQ ID NO: 110) ASISCRSSQSLVHSNGNTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTNFTLKISRVEAEDLGLYFCS.

Anti-Langerin-2G3H-LV-hIgG4H-C, heavy chain, SEQ ID NO:111:

(SEQ ID NO: 111) MTLNMLLGLRWVFFVVFYQGVHCEVQLVESGGGLVQPKGSLKLSCAASGLTFNIYAMNWVRQAPGKGLEWVARIRNKSNNYATYYADSVKDRFTISRDDSQSLLYLQMNNLKTEDTAMYYCVGRDWFDYWGQGTLVTVSAAKTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKAS.

The H chain variable of the Ab 2G3 is shown in SEQ ID NO:112:

(SEQ ID NO: 112) SLKLSCAASGLTFNIYAMNWVRQAPGKGLEWVARIRNKSNNYATYYADSVKDRFTISRDDSQSLLYLQMNNLKTEDTAMYYC.

Anti-Langerin-2G3L-LV-hIgGK-C, light chain, SEQ ID NO:113:

(SEQ ID NO: 113) MAWISLILSLLALSSGAISQAVVTQESALTTSPGETVTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTNNRVSGVPARFSGSLIGDKAALTITGAQTEDEAIYFCALWYSNHWVFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

The L chain variable of the Ab 2G3 is shown in SEQ ID NO:114:

(SEQ ID NO: 114) VTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTNNRVSGVPARFSGSLIGDKAALTITGAQTEDEAIYFCA.

III. INTERLEUKIN 10 (IL-10)

Interleukin-10 (IL-10), also known as human cytokine synthesisinhibitory factor (CSIF), is an anti-inflammatory cytokine. In humans,IL-10 is encoded by the IL10 gene. The mRNA sequence of human IL-10 isrepresented by accession No.: NM_000572.2. The amino acid sequence ofhuman IL-10 is represented by accession No.: NP_000563.1 and SEQ IDNO:5. The sequence associated with these accession numbers isincorporated by reference for all purposes.

In some embodiments, the APC-targeted antibody or fragment thereof isoperatively linked to an IL-10 polypeptide comprising a sequencecorresponding to a protein sequence of an NCBI accession numberNP_000563.1. In some embodiments, the IL-10 polypeptide comprises theamino acid sequence of SEQ ID NO:5 or a fragment thereof:

IV. ANTIGENS

Certain aspects of the disclosure include methods and compositionsconcerning antigenic components including segments, fragments, orepitopes of polypeptides, peptides, nucleic acids, carbohydrates, lipidsand other molecules that provoke or induce an antigenic response,generally referred to as antigens. In one embodiment, the antigen is apeptide. In particular, antigens, or antigenic segments or fragments ofsuch antigens, which lead to the destruction of a cell via an immuneresponse, can be identified and used in the methods and compositionsdescribed herein.

Antigens associated with various diseases and disorders are known in theart. It is contemplated that any antigen may be used in the methods andcompositions described herein. In certain aspects, the antigen is onethat is involved in the etiology of an autoimmune, allergic, orinflammatory disease known in the art and/or described herein.

In certain aspects, the antigen is one known in the art to be involvedin rheumatoid arthritis, allergy, asthma, systemic onset juvenilearthritis, inflammatory bowel disease, systemic lupus erythematosus,type 1 diabetes, and Crohn's disease.

V. PEPTIDE COMPONENTS AND PROTEINACEOUS COMPOSITIONS

Polypeptides and peptides may be modified by various amino aciddeletions, insertions, and/or substitutions. In particular embodiments,modified polypeptides and/or peptides are capable of modulating animmune response in a subject. As used herein, a “protein” or“polypeptide” or “peptide” refers to a molecule comprising at least fiveamino acid residues. In some embodiments, a wild-type version of aprotein or peptide are employed, however, in many embodiments, amodified protein or polypeptide is employed to generate the antibodyconjugates described herein. A “modified protein” or “modifiedpolypeptide” or “modified peptide” refers to a protein or polypeptidewhose chemical structure, particularly its amino acid sequence, isaltered with respect to the wild-type protein or polypeptide.

Peptides include peptides that are found to be specific to cancerous orpre-cancerous cells in the body. These peptides may be associated withthe APC-targeted antibodies described herein. Administration ofcombinations of these peptides includes administering a population ofantibody conjugates having multiple peptides attached and/oradministering multiple conjugate populations, each having a specificpeptide attached or a combination of such conjugates that includesnanoparticles with 1, 2, 3, 4, 5, 6, or more peptides attached to theAPC-targeted antibody, antigen binding fragment thereof, or IL-10protein.

Proteinaceous compositions may be made by any technique known to thoseof skill in the art, including (i) the expression of proteins,polypeptides, or peptides through standard molecular biologicaltechniques, (ii) the isolation of proteinaceous compounds from naturalsources, or (iii) the chemical synthesis of proteinaceous materials. Thenucleotide as well as the protein, polypeptide, and peptide sequencesfor various genes have been previously disclosed, and may be found inthe recognized computerized databases. One such database is the NationalCenter for Biotechnology Information's GenBank and GenPept databases (onthe World Wide Web at ncbi.nlm.nih.gov/). The all or part of the codingregions for these genes may be amplified and/or expressed using thetechniques disclosed herein or as would be known to those of ordinaryskill in the art.

Amino acid sequence variants of antigenic epitopes and otherpolypeptides of these compositions can be substitutional, insertional,or deletion variants. A modification in a polypeptide may affect 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 98, 99, 100, 100, 101, 102, 103, 104, 105, 106, 107, 108,109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122,123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136,137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150,151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164,165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178,179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192,193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206,207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220,221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234,235, 236, 237, 238, 239, 240, 241, 242, 235, 236, 237, 238, 239, 240,241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254,255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268,269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282,283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296,297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310,311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324,325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338,339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352,353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366,367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380,381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394,395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408,409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422,423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436,437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450,451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464,465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478,479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492,493, 494, 495, 496, 497, 498, 499, 500 or more non-contiguous orcontiguous amino acids of a peptide or polypeptide, as compared towild-type. A peptide or polypeptide that results in an immune responseis contemplated for use in embodiments.

Deletion variants typically lack one or more residues of the native orwild-type amino acid sequence. Individual residues can be deleted or anumber of contiguous amino acids can be deleted. A stop codon may beintroduced (by substitution or insertion) into an encoding nucleic acidsequence to generate a truncated protein. Insertional mutants typicallyinvolve the addition of material at a non-terminal point in thepolypeptide. This may include the insertion of one or more residues.Terminal additions, called fusion proteins, may also be generated.

Substitutional variants typically contain the exchange of one amino acidfor another at one or more sites within the protein, and may be designedto modulate one or more properties of the polypeptide, with or withoutthe loss of other functions or properties. Substitutions may beconservative, that is, one amino acid is replaced with one of similarshape and charge. Conservative substitutions are well known in the artand include, for example, the changes of: alanine to serine; arginine tolysine; asparagine to glutamine or histidine; aspartate to glutamate;cysteine to serine; glutamine to asparagine; glutamate to aspartate;glycine to proline; histidine to asparagine or glutamine; isoleucine toleucine or valine; leucine to valine or isoleucine; lysine to arginine;methionine to leucine or isoleucine; phenylalanine to tyrosine, leucineor methionine; serine to threonine; threonine to serine; tryptophan totyrosine; tyrosine to tryptophan or phenylalanine; and valine toisoleucine or leucine. Alternatively, substitutions may benon-conservative such that a function or activity of a polypeptide orpeptide is affected, such as avidity or affinity for a cellularreceptor(s). Non-conservative changes typically involve substituting aresidue with one that is chemically dissimilar, such as a polar orcharged amino acid for a nonpolar or uncharged amino acid, and viceversa.

Proteins may be recombinant, or synthesized in vitro. Alternatively, arecombinant protein may be isolated from bacteria or other host cell.

The term “functionally equivalent codon” is used herein to refer tocodons that encode the same amino acid, such as the six codons forarginine or serine, and also refers to codons that encode biologicallyequivalent amino acids.

It also will be understood that amino acid and nucleic acid sequencesmay include additional residues, such as additional N- or C-terminalamino acids, or 5′ or 3′ nucleic acid sequences, respectively, and yetstill be essentially as set forth in one of the sequences disclosedherein, so long as the sequence meets the criteria set forth above,including the maintenance of biological protein activity (e.g.,immunogenicity). The addition of terminal sequences particularly appliesto nucleic acid sequences that may, for example, include variousnon-coding sequences flanking either of the 5′ or 3′ portions of thecoding region.

It is contemplated that in composition embodiments, there is betweenabout 0.001 mg and about 10 mg of total protein per ml. Thus, theconcentration of protein in a composition can be about, at least aboutor at most about 0.001, 0.010, 0.050, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7,0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5,7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 50, 100 .mu.g/ml or mg/ml or more(or any range derivable therein).

Embodiments include in some cases the administration of an APC-targetedantibody. In some embodiments, the methods and compositions furthercomprise an antigen. U.S. Pat. No. 4,554,101 (Hopp), which isincorporated herein by reference, teaches the identification andpreparation of antigenic epitopes from primary amino acid sequences onthe basis of hydrophilicity. Through the methods disclosed in Hopp, oneof skill in the art would be able to identify potential antigenicepitopes from within an amino acid sequence and confirm theirimmunogenicity. Numerous scientific publications have also been devotedto the prediction of secondary structure and to the identification ofepitopes, from analyses of amino acid sequences (Chou & Fasman, 1974a,b;1978a,b; 1979). Any of these may be used, if desired, to supplement theteachings of Hopp in U.S. Pat. No. 4,554,101.

VI. PHARMACEUTICAL COMPOSITIONS

Embodiments include methods and compositions for increasing immuneresponses in a subject in need thereof. They include compositions thatcan be used to induce or modify an immune response against an antigene.g., a polypeptide, a peptide, a carbohydrate, a lipid or othermolecule or molecular fragment and against developing a condition ordisease associated with such antigen.

It is contemplated that the APC-targeted antibody or antigen bindingfragment thereof (and optionally antigen and optionally linked to IL-10)may be administered with additional adjuvants known in the art such asTLR agonists. TLR agonists may include an agonist to TLR1 (e.g.peptidoglycan or triacyl lipoproteins), TLR2 (e.g. lipoteichoic acid;peptidoglycan from Bacillus subtilis, E. coli 0111:B4, Escherichia coliK12, or Staphylococcus aureus; atypical lipopolysaccharide (LPS) such asLeptospirosis LPS and Porphyromonas gingivalis LPS; a syntheticdiacylated lipoprotein such as FSL-1 or Pam2CSK4; lipoarabinomannan orlipomannan from M. smegmatis; triacylated lipoproteins such as Pam3CSK4;lipoproteins such as MALP-2 and MALP-404 from mycoplasma; Borreliaburgdorferi OspA; Porin from Neisseria meningitidis or Haemophilusinfluenza; Propionibacterium acnes antigen mixtures; Yersinia LcrV;lipomannan from Mycobacterium or Mycobacterium tuberculosis; Trypanosomacruzi GPI anchor; Schistosoma mansoni lysophosphatidylserine; Leishmaniamajor lipophosphoglycan (LPG); Plasmodium falciparumglycophosphatidylinositol (GPI); zymosan; antigen mixtures fromAspergillus fumigatus or Candida albicans; and measles hemagglutinin),TLR3 (e.g. double-stramded RNA, polyadenylic-polyuridylic acid(Poly(A:U)); polyinosine-polycytidylic acid (Poly(I:C));polyinosine-polycytidylic acid high molecular weight (Poly(I:C) HMW);and polyinosine-polycytidylic acid low molecular weight (Poly(I:C)LMW)), TLR4 (e.g. LPS from Escherichia coli and Salmonella species);TLR5 (e.g. Flagellin from B. subtilis, P. aeruginosa, or S.typhimurium), TLR8 (e.g. single stranded RNAs such as ssRNA with 6UUAUrepeats, RNA homopolymer (ssPolyU naked), HIV-1 LTR-derived ssRNA(ssRNA40), or ssRNA with 2 GUCCUUCAA repeats (ssRNA-DR)), TLR7 (e.g.imidazoquinoline compound imiquimod, Imiquimod VacciGrade™, GardiquimodVacciGrade™, or Gardiquimod™; adenine analog CL264; base analog CL307;guanosine analog loxoribine; TLR7/8 (e.g. thiazoquinoline compoundCL075; imidazoquinoline compound CL097, R848, or R848 VacciGrade™), TLR9(e.g. CpG ODNs); and TLR11 (e.g. Toxoplasma gondii Profilin). In certainembodiments, the TLR agonist is a specific agonist listed above. Infurther embodiments, the TLR agonist is one that agonizes either one TLRor two TLRs specifically.

In certain embodiments, the methods and compositions specificallyexclude the administration of a TLR ligand and/or agonist.

Administration of the compositions will typically be via any commonroute. This includes, but is not limited to oral, parenteral,orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal,intranasal, by inhalation, by using a nebulizer, or by intravenousinjection. In certain embodiments, a vaccine composition may be inhaled(e.g., U.S. Pat. No. 6,651,655, which is specifically incorporated byreference). Additional formulations which are suitable for other modesof administration include oral formulations. Oral formulations includesuch normally employed excipients as, for example, pharmaceutical gradesof mannitol, lactose, starch, magnesium stearate, sodium saccharine,cellulose, magnesium carbonate and the like. These compositions take theform of solutions, suspensions, tablets, pills, capsules, sustainedrelease formulations or powders and contain about 10% to about 95% ofactive ingredient, preferably about 25% to about 70%.

Typically, compositions are administered in a manner compatible with thedosage formulation, and in such amount as will be therapeuticallyeffective and immune modifying. The quantity to be administered dependson the subject to be treated. Precise amounts of active ingredientrequired to be administered depend on the judgment of the practitioner.

The manner of application may be varied widely. Any of the conventionalmethods for administration of an antibody are applicable. These arebelieved to include oral application on a solid physiologicallyacceptable base or in a physiologically acceptable dispersion,parenterally, by injection and the like. The dosage of thepharmaceutical composition will depend on the route of administrationand will vary according to the size and health of the subject.

In many instances, it will be desirable to have multiple administrationsof at most about or at least about 3, 4, 5, 6, 7, 8, 9, 10 or more. Theadministrations may range from 2 day to twelve week intervals, moreusually from one to two week intervals. The course of theadministrations may be followed by assays for reactive immune responsesand T cell activity.

The phrases “pharmaceutically acceptable” or “pharmacologicallyacceptable” refer to molecular entities and compositions that do notproduce an adverse, allergic, or other untoward reaction whenadministered to an animal, or human. As used herein, “pharmaceuticallyacceptable carrier” includes any and all solvents, dispersion media,coatings, antibacterial and antifungal agents, isotonic and absorptiondelaying agents, and the like. The use of such media and agents forpharmaceutical active substances is well known in the art. Exceptinsofar as any conventional media or agent is incompatible with theactive ingredients, its use in immunogenic and therapeutic compositionsis contemplated.

The antibodies or antigen binding fragments can be formulated forparenteral administration, e.g., formulated for injection via theintravenous, intradermal, intramuscular, sub-cutaneous, or evenintraperitoneal routes. In a specific embodiment, the composition isadministered by intradermal injection. In further embodiments, thecomposition is administered by intravenous injection. The preparation ofan aqueous composition that contains a APC-targeted antibody thatmodifies the subject's immune condition will be known to those of skillin the art in light of the present disclosure. Typically, suchcompositions can be prepared as injectables, either as liquid solutionsor suspensions; solid forms suitable for use to prepare solutions orsuspensions upon the addition of a liquid prior to injection can also beprepared; and, the preparations can also be emulsified.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions; formulations including sesame oil,peanut oil, or aqueous propylene glycol; and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases the form must be sterile and must be fluid tothe extent that it may be easily injected. It also should be stableunder the conditions of manufacture and storage and must be preservedagainst the contaminating action of microorganisms, such as bacteria andfungi.

The compositions may be formulated into a neutral or salt form.Pharmaceutically acceptable salts, include the acid addition salts(formed with the free amino groups of the protein) and which are formedwith inorganic acids such as, for example, hydrochloric or phosphoricacids, or such organic acids as acetic, oxalic, tartaric, mandelic, andthe like. Salts formed with the free carboxyl groups can also be derivedfrom inorganic bases such as, for example, sodium, potassium, ammonium,calcium, or ferric hydroxides, and such organic bases as isopropylamine,trimethylamine, histidine, procaine and the like.

The carrier can also be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyethylene glycol, and the like), suitable mixturesthereof, and vegetable oils. The prevention of the action ofmicroorganisms can be brought about by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars or sodium chloride.Prolonged absorption of the injectable compositions can be brought aboutby the use in the compositions of agents delaying absorption, forexample, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activeingredients in the required amount in the appropriate solvent withvarious of the other ingredients enumerated above, as required, followedby filtered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques, which yield a powder of the active ingredient, plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

An effective amount of therapeutic or prophylactic composition isdetermined based on the intended goal. The term “unit dose” or “dosage”refers to physically discrete units suitable for use in a subject, eachunit containing a predetermined quantity of the composition calculatedto produce the desired responses discussed above in association with itsadministration, i.e., the appropriate route and regimen. The quantity tobe administered, both according to number of treatments and unit dose,depends on the result and/or protection desired. Precise amounts of thecomposition also depend on the judgment of the practitioner and arepeculiar to each individual. Factors affecting dose include physical andclinical state of the subject, route of administration, intended goal oftreatment (alleviation of symptoms versus cure), and potency, stability,and toxicity of the particular composition. Upon formulation, solutionswill be administered in a manner compatible with the dosage formulationand in such amount as is therapeutically or prophylactically effective.The formulations are easily administered in a variety of dosage forms,such as the type of injectable solutions described above.

VII. IN VITRO OR EX VIVO ADMINISTRATION

As used herein, the term in vitro administration refers to manipulationsperformed on cells removed from or outside of a subject, including, butnot limited to cells in culture. The term ex vivo administration refersto cells which have been manipulated in vitro, and are subsequentlyadministered to a subject. The term in vivo administration includes allmanipulations performed within a subject, including administrations.

In certain aspects, the compositions may be administered either invitro, ex vivo, or in vivo. In certain in vitro embodiments, isolatedimmune cells are incubated with compositions described herein. Forexample, isolated APCs may be incubated with the antibody or antibodyconjugates as described herein. The cells can then be used for in vitroanalysis, or alternatively for ex vivo administration.

VIII. THERAPEUTIC APPLICATIONS

Methods include treatment of inflammatory and autoimmune disordersMethods may be employed with respect to individuals who has testedpositive for such disorders or who are deemed to be at risk fordeveloping such a condition or related condition.

The antibody or antigen binding fragment of the disclosure (in someembodiments, conjugated to IL-10) can be given to induce or modify animmune response in a person having, suspected of having, or at risk ofdeveloping an autoimmune condition or complication relating to anallograft. Methods may be employed with respect to individuals who havetested positive for autoreactivity or allo-reactivity or who are deemedto be at risk for developing such a condition or related condition.

The methods described herein are particularly useful in treating orpreventing disorders for which antigenic determinants are poorlycharacterized. Such disorders include, for example, rheumatoidarthritis, allergy, asthma, systemic onset juvenile arthritis,inflammatory bowel disease, and Crohn's disease. The methods describedherein are also particularly useful for disorders such as GVHD and graftrejection since the antigenic determinants of such diseases may not beknown or may be different depending on the tissue and/or individual fromwhich the tissue was obtained from.

It is contemplated that targeting dendritic cells (e.g. with ananti-DC-ASGPR antibody) inhibits autoimmune diseases but does notinterfere with pathogen-specific T cell responses.

Embodiments can be used to treat or ameliorate a number ofimmune-mediated, inflammatory, or autoimmune-inflammatory diseases,e.g., allergies, asthma, diabetes (e.g. type 1 diabetes), graftrejection, etc. Examples of such diseases or disorders also include, butare not limited to arthritis (rheumatoid arthritis such as acutearthritis, chronic rheumatoid arthritis, gout or gouty arthritis, acutegouty arthritis, acute immunological arthritis, chronic inflammatoryarthritis, degenerative arthritis, type II collagen-induced arthritis,infectious arthritis, Lyme arthritis, proliferative arthritis, psoriaticarthritis, Still's disease, vertebral arthritis, and systemicjuvenile-onset rheumatoid arthritis, osteoarthritis, arthritis chronicaprogrediente, arthritis deformans, polyarthritis chronica primaria,reactive arthritis, and ankylosing spondylitis), inflammatoryhyperproliferative skin diseases, psoriasis such as plaque psoriasis,gutatte psoriasis, pustular psoriasis, and psoriasis of the nails, atopyincluding atopic diseases such as hay fever and Job's syndrome,dermatitis including contact dermatitis, chronic contact dermatitis,exfoliative dermatitis, allergic dermatitis, allergic contactdermatitis, dermatitis herpetiformis, nummular dermatitis, seborrheicdermatitis, non-specific dermatitis, primary irritant contactdermatitis, and atopic dermatitis, x-linked hyper IgM syndrome, allergicintraocular inflammatory diseases, urticaria such as chronic allergicurticaria and chronic idiopathic urticaria, including chronic autoimmuneurticaria, myositis, polymyositis/dermatomyositis, juveniledermatomyositis, toxic epidermal necrolysis, scleroderma (includingsystemic scleroderma), sclerosis such as systemic sclerosis, multiplesclerosis (MS) such as spino-optical MS, primary progressive MS (PPMS),and relapsing remitting MS (RRMS), progressive systemic sclerosis,atherosclerosis, arteriosclerosis, sclerosis disseminata, ataxicsclerosis, neuromyelitis optica (NMO), inflammatory bowel disease (IBD)(for example, Crohn's disease, autoimmune-mediated gastrointestinaldiseases, colitis such as ulcerative colitis, colitis ulcerosa,microscopic colitis, collagenous colitis, colitis polyposa, necrotizingenterocolitis, and transmural colitis, and autoimmune inflammatory boweldisease), bowel inflammation, pyoderma gangrenosum, erythema nodosum,primary sclerosing cholangitis, respiratory distress syndrome, includingadult or acute respiratory distress syndrome (ARDS), meningitis,inflammation of all or part of the uvea, iritis, choroiditis, anautoimmune hematological disorder, rheumatoid spondylitis, rheumatoidsynovitis, hereditary angioedema, cranial nerve damage as in meningitis,herpes gestationis, pemphigoid gestationis, pruritis scroti, autoimmunepremature ovarian failure, sudden hearing loss due to an autoimmunecondition, IgE-mediated diseases such as anaphylaxis and allergic andatopic rhinitis, encephalitis such as Rasmussen's encephalitis andlimbic and/or brainstem encephalitis, uveitis, such as anterior uveitis,acute anterior uveitis, granulomatous uveitis, nongranulomatous uveitis,phacoantigenic uveitis, posterior uveitis, or autoimmune uveitis,glomerulonephritis (GN) with and without nephrotic syndrome such aschronic or acute glomerulonephritis such as primary GN, immune-mediatedGN, membranous GN (membranous nephropathy), idiopathic membranous GN oridiopathic membranous nephropathy, membrano- or membranous proliferativeGN (MPGN), including Type I and Type II, and rapidly progressive GN,proliferative nephritis, autoimmune polyglandular endocrine failure,balanitis including balanitis circumscripta plasmacellularis,balanoposthitis, erythema annulare centrifugum, erythema dyschromicumperstans, eythema multiform, granuloma annulare, lichen nitidus, lichensclerosus et atrophicus, lichen simplex chronicus, lichen spinulosus,lichen planus, lamellar ichthyosis, epidermolytic hyperkeratosis,premalignant keratosis, pyoderma gangrenosum, allergic conditions andresponses, allergic reaction, eczema including allergic or atopiceczema, asteatotic eczema, dyshidrotic eczema, and vesicularpalmoplantar eczema, asthma such as asthma bronchiale, bronchial asthma,and auto-immune asthma, conditions involving infiltration of T cells andchronic inflammatory responses, immune reactions against foreignantigens such as fetal A-B-O blood groups during pregnancy, chronicpulmonary inflammatory disease, autoimmune myocarditis, leukocyteadhesion deficiency, lupus, including lupus nephritis, lupus cerebritis,pediatric lupus, non-renal lupus, extra-renal lupus, discoid lupus anddiscoid lupus erythematosus, alopecia lupus, systemic lupuserythematosus (SLE) such as cutaneous SLE or subacute cutaneous SLE,neonatal lupus syndrome (NLE), and lupus erythematosus disseminatus,juvenile onset (Type I) diabetes mellitus, including pediatricinsulin-dependent diabetes mellitus (IDDM), and adult onset diabetesmellitus (Type II diabetes) and autoimmune diabetes. Also contemplatedare immune responses associated with acute and delayed hypersensitivitymediated by cytokines and T-lymphocytes, sarcoidosis, granulomatosisincluding lymphomatoid granulomatosis, Wegener's granulomatosis,agranulocytosis, vasculitides, including vasculitis, large-vesselvasculitis (including polymyalgia rheumatica and gianT cell (Takayasu's)arteritis), medium-vessel vasculitis (including Kawasaki's disease andpolyarteritis nodosa/periarteritis nodosa), microscopic polyarteritis,immunovasculitis, CNS vasculitis, cutaneous vasculitis, hypersensitivityvasculitis, necrotizing vasculitis such as systemic necrotizingvasculitis, and ANCA-associated vasculitis, such as Churg-Straussvasculitis or syndrome (CSS) and ANCA-associated small-vesselvasculitis, temporal arteritis, aplastic anemia, autoimmune aplasticanemia, Coombs positive anemia, Diamond Blackfan anemia, hemolyticanemia or immune hemolytic anemia including autoimmune hemolytic anemia(AIHA), Addison's disease, autoimmune neutropenia, pancytopenia,leukopenia, diseases involving leukocyte diapedesis, CNS inflammatorydisorders, Alzheimer's disease, Parkinson's disease, multiple organinjury syndrome such as those secondary to septicemia, trauma orhemorrhage, antigen-antibody complex-mediated diseases, anti-glomerularbasement membrane disease, anti-phospholipid antibody syndrome, allergicneuritis, Behcet's disease/syndrome, Castleman's syndrome, Goodpasture'ssyndrome, Reynaud's syndrome, Sjogren's syndrome, Stevens-Johnsonsyndrome, pemphigoid such as pemphigoid bullous and skin pemphigoid,pemphigus (including pemphigus vulgaris, pemphigus foliaceus, pemphigusmucus-membrane pemphigoid, and pemphigus erythematosus), autoimmunepolyendocrinopathies, Reiter's disease or syndrome, thermal injury,preeclampsia, an immune complex disorder such as immune complexnephritis, antibody-mediated nephritis, polyneuropathies, chronicneuropathy such as IgM polyneuropathies or IgM-mediated neuropathy,autoimmune or immune-mediated thrombocytopenia such as idiopathicthrombocytopenic purpura (ITP) including chronic or acute ITP, scleritissuch as idiopathic cerato-scleritis, episcleritis, autoimmune disease ofthe testis and ovary including autoimmune orchitis and oophoritis,primary hypothyroidism, hypoparathyroidism, autoimmune endocrinediseases including thyroiditis such as autoimmune thyroiditis,Hashimoto's disease, chronic thyroiditis (Hashimoto's thyroiditis), orsubacute thyroiditis, autoimmune thyroid disease, idiopathichypothyroidism, Grave's disease, polyglandular syndromes such asautoimmune polyglandular syndromes (or polyglandular endocrinopathysyndromes), paraneoplastic syndromes, including neurologicparaneoplastic syndromes such as Lambert-Eaton myasthenic syndrome orEaton-Lambert syndrome, stiff-man or stiff-person syndrome,encephalomyelitis such as allergic encephalomyelitis orencephalomyelitis allergica and experimental allergic encephalomyelitis(EAE), experimental autoimmune encephalomyelitis, myasthenia gravis suchas thymoma-associated myasthenia gravis, cerebellar degeneration,neuromyotonia, opsoclonus or opsoclonus myoclonus syndrome (OMS), andsensory neuropathy, multifocal motor neuropathy, Sheehan's syndrome,autoimmune hepatitis, chronic hepatitis, lupoid hepatitis, gianT cellhepatitis, chronic active hepatitis or autoimmune chronic activehepatitis, lymphoid interstitial pneumonitis (LIP), bronchiolitisobliterans (non-transplant) vs NSIP, Guillain-Barre syndrome, Berger'sdisease (IgA nephropathy), idiopathic IgA nephropathy, linear IgAdermatosis, acute febrile neutrophilic dermatosis, subcorneal pustulardermatosis, transient acantholytic dermatosis, cirrhosis such as primarybiliary cirrhosis and pneumonocirrhosis, autoimmune enteropathysyndrome, Celiac or Coeliac disease, celiac sprue (gluten enteropathy),refractory sprue, idiopathic sprue, cryoglobulinemia, amylotrophiclateral sclerosis (ALS; Lou Gehrig's disease), coronary artery disease,autoimmune ear disease such as autoimmune inner ear disease (AIED),autoimmune hearing loss, polychondritis such as refractory or relapsedor relapsing polychondritis, pulmonary alveolar proteinosis, Cogan'ssyndrome/nonsyphilitic interstitial keratitis, Bell's palsy, Sweet'sdisease/syndrome, rosacea autoimmune, zoster-associated pain,amyloidosis, a non-cancerous lymphocytosis, a primary lymphocytosis,which includes monoclonal B cell lymphocytosis (e.g., benign monoclonalgammopathy and monoclonal gammopathy of undetermined significance,MGUS), peripheral neuropathy, paraneoplastic syndrome, channelopathiessuch as epilepsy, migraine, arrhythmia, muscular disorders, deafness,blindness, periodic paralysis, and channelopathies of the CNS, autism,inflammatory myopathy, focal or segmental or focal segmentalglomerulosclerosis (FSGS), endocrine opthalmopathy, uveoretinitis,chorioretinitis, autoimmune hepatological disorder, fibromyalgia,multiple endocrine failure, Schmidt's syndrome, adrenalitis, gastricatrophy, presenile dementia, demyelinating diseases such as autoimmunedemyelinating diseases and chronic inflammatory demyelinatingpolyneuropathy, Dressler's syndrome, alopecia greata, alopecia totalis,CREST syndrome (calcinosis, Raynaud's phenomenon, esophagealdysmotility, sclerodactyl), and telangiectasia), male and femaleautoimmune infertility, e.g., due to anti-spermatozoan antibodies, mixedconnective tissue disease, Chagas' disease, rheumatic fever, recurrentabortion, farmer's lung, erythema multiforme, post-cardiotomy syndrome,Cushing's syndrome, bird-fancier's lung, allergic granulomatousangiitis, benign lymphocytic angiitis, Alport's syndrome, alveolitissuch as allergic alveolitis and fibrosing alveolitis, interstitial lungdisease, transfusion reaction, leprosy, malaria, parasitic diseases suchas leishmaniasis, kypanosomiasis, schistosomiasis, ascariasis,aspergillosis, Sampter's syndrome, Caplan's syndrome, dengue,endocarditis, endomyocardial fibrosis, diffuse interstitial pulmonaryfibrosis, interstitial lung fibrosis, pulmonary fibrosis, idiopathicpulmonary fibrosis, cystic fibrosis, endophthalmitis, erythema elevatumet diutinum, erythroblastosis fetalis, eosinophilic faciitis, Shulman'ssyndrome, Felty's syndrome, flariasis, cyclitis such as chroniccyclitis, heterochronic cyclitis, iridocyclitis (acute or chronic), orFuch's cyclitis, Henoch-Schonlein purpura, human immunodeficiency virus(HIV) infection, SCID, acquired immune deficiency syndrome (AIDS),echovirus infection, sepsis, endotoxemia, pancreatitis, thyroxicosis,parvovirus infection, rubella virus infection, post-vaccinationsyndromes, congenital rubella infection, Epstein-Barr virus infection,mumps, Evan's syndrome, autoimmune gonadal failure, Sydenham's chorea,post-streptococcal nephritis, thromboangitis ubiterans, thyrotoxicosis,tabes dorsalis, chorioiditis, gianT cell polymyalgia, chronichypersensitivity pneumonitis, keratoconjunctivitis sicca, epidemickeratoconjunctivitis, idiopathic nephritic syndrome, minimal changenephropathy, benign familial and ischemia-reperfusion injury, transplantorgan reperfusion, retinal autoimmunity, joint inflammation, bronchitis,chronic obstructive airway/pulmonary disease, silicosis, aphthae,aphthous stomatitis, arteriosclerotic disorders, asperniogenese,autoimmune hemolysis, Boeck's disease, cryoglobulinemia, Dupuytren'scontracture, endophthalmia phacoanaphylactica, enteritis allergica,erythema nodosum leprosum, idiopathic facial paralysis, chronic fatiguesyndrome, febris rheumatica, Hamman-Rich's disease, sensoneural hearingloss, haemoglobinuria paroxysmatica, hypogonadism, ileitis regionalis,leucopenia, mononucleosis infectiosa, traverse myelitis, primaryidiopathic myxedema, nephrosis, ophthalmia symphatica, orchitisgranulomatosa, pancreatitis, polyradiculitis acuta, pyodermagangrenosum, Quervain's thyreoiditis, acquired spenic atrophy,non-malignant thymoma, vitiligo, toxic-shock syndrome, food poisoning,conditions involving infiltration of T cells, leukocyte-adhesiondeficiency, immune responses associated with acute and delayedhypersensitivity mediated by cytokines and T-lymphocytes, diseasesinvolving leukocyte diapedesis, multiple organ injury syndrome,antigen-antibody complex-mediated diseases, antiglomerular basementmembrane disease, allergic neuritis, autoimmune polyendocrinopathies,oophoritis, primary myxedema, autoimmune atrophic gastritis, sympatheticophthalmia, rheumatic diseases, mixed connective tissue disease,nephrotic syndrome, insulitis, polyendocrine failure, autoimmunepolyglandular syndrome type I, adult-onset idiopathic hypoparathyroidism(AOIH), cardiomyopathy such as dilated cardiomyopathy, epidermolisisbullosa acquisita (EBA), hemochromatosis, myocarditis, nephroticsyndrome, primary sclerosing cholangitis, purulent or nonpurulentsinusitis, acute or chronic sinusitis, ethmoid, frontal, maxillary, orsphenoid sinusitis, an eosinophil-related disorder such as eosinophilia,pulmonary infiltration eosinophilia, eosinophilia-myalgia syndrome,Loffler's syndrome, chronic eosinophilic pneumonia, tropical pulmonaryeosinophilia, bronchopneumonic aspergillosis, aspergilloma, orgranulomas containing eosinophils, anaphylaxis, seronegativespondyloarthritides, polyendocrine autoimmune disease, sclerosingcholangitis, sclera, episclera, chronic mucocutaneous candidiasis,Bruton's syndrome, transient hypogammaglobulinemia of infancy,Wiskott-Aldrich syndrome, ataxia telangiectasia syndrome, angiectasis,autoimmune disorders associated with collagen disease, rheumatism,neurological disease, lymphadenitis, reduction in blood pressureresponse, vascular dysfunction, tissue injury, cardiovascular ischemia,hyperalgesia, renal ischemia, cerebral ischemia, and diseaseaccompanying vascularization, allergic hypersensitivity disorders,glomerulonephritides, reperfusion injury, ischemic re-perfusiondisorder, reperfusion injury of myocardial or other tissues,lymphomatous tracheobronchitis, inflammatory dermatoses, dermatoses withacute inflammatory components, multiple organ failure, bullous diseases,renal cortical necrosis, acute purulent meningitis or other centralnervous system inflammatory disorders, ocular and orbital inflammatorydisorders, granulocyte transfusion-associated syndromes,cytokine-induced toxicity, narcolepsy, acute serious inflammation,chronic intractable inflammation, pyelitis, endarterial hyperplasia,peptic ulcer, valvulitis, graft versus host disease, contacthypersensitivity, asthmatic airway hyperreaction, and endometriosis.

Embodiments can be used to prevent, treat or ameliorate a number ofallergic disorders. Non-limiting examples include asthma, type 1diabetes, chronic obstructive pulmonary disease, interstitial lungdisease, chronic obstructive lung disease, chronic bronchitis,eosinophilic bronchitis, eosinophilic pneumonia, pneumonia, inflammatorybowel disease, atopic dermatitis, atopy, allergy, allergic rhinitis,idiopathic pulmonary fibrosis, scleroderma, emphysema, breast cancer,and ulcerative colitis. Non-limiting examples of allergic disordersinclude allergic atopy and dermatitis, allergic rhinitis, allergicasthma, allergic responses to food (e.g. milk, egg, wheat, nut, fish,shellfish, sulfite, soy, and casein), environmental allergens (e.g.plant and animal allergens such as dander, dust mites, pollen, cedar,poison ivy, poison oak, poison sumac, etc. . . . ), insect bites (e.g.bee, wasp, yellow jacket, hornet, or fire ant stings), hay fever,allergic conjunctivitis, hives, mold, medication allergies (e.g. aspirinand penicillin), and cosmetic allergies.

In some embodiments, the compositions and methods described herein areused to treat an inflammatory component of a disorder listed hereinand/or known in the art. Accordingly, the methods and compositionsdescribed herein can be used to treat a subject suffering frominflammation. In some embodiments, the inflammation is acute. In otherembodiments, the inflammation is chronic. In further embodiments, thecompositions and methods described herein are used to treat or prevent acancer by treating or preventing an inflammatory component associatedwith the cancer. In some embodiments, the cancer is breast cancer.

IX. COMBINATION THERAPY

The compositions and related methods disclosed herein, particularlyadministration of an APC-targeted antibody or antigen binding fragmentmay also be used in combination with the administration of traditionaltherapies. These include, but are not limited to, the administration ofimmunosuppressive or modulating therapies or treatments. Non-limitingexamples of existing immunosuppressive therapies include administrationof immunosuppressive compounds such as cyclosporine A, cyclophosphamide,FK506, tacrolimus, corticosteroids, azathioprine, mycophenolate mofetil,sirolimus, rapamycin, rapamycin analogs, deoxyspergualin, and prednisone

In one aspect, it is contemplated that an APC-targeted antibody orantigen binding fragment is used in conjunction with a cytokinetreatment. Alternatively, antibody administration may precede or followthe other treatment by intervals ranging from minutes to weeks. Inembodiments where the other agents are administered separately, onewould generally ensure that a significant period of time did not expirebetween the time of each delivery, such that the agent and antibodywould still be able to exert an advantageously combined effect on thesubject. In such instances, it is contemplated that one may administerboth modalities within about 12-24 h of each other and, more preferably,within about 6-12 h of each other. In some situations, it may bedesirable to extend the time period for administration significantly,however, where several days (2, 3, 4, 5, 6 or 7) to several weeks (1, 2,3, 4, 5, 6, 7 or 8) lapse between the respective administrations.

Administration of the anti-DC-ASGPR antibody or antigen binding fragmentcompositions to a patient/subject will follow general protocols for theadministration of such compounds, taking into account the toxicity, ifany. It is expected that the treatment cycles would be repeated asnecessary. It also is contemplated that various standard therapies, suchas hydration, may be applied in combination with the described therapy.

X. EXAMPLES

The following examples are included to demonstrate certain embodiments.It should be appreciated by those of skill in the art that thetechniques disclosed in the examples which follow represent techniquesdiscovered by the inventor to function well in the practice of theinvention, and thus can be considered to constitute preferred modes forits practice. However, those of skill in the art should, in light of thepresent disclosure, appreciate that many changes can be made in thespecific embodiments which are disclosed and still obtain a like orsimilar result without departing from the spirit and scope of theinvention.

Example 1: Targeting IL-10 to Antigen Presenting Cells

With targeted in vivo delivery of IL-10 to APCs followed by alterationsof pathogenic functions of APC as well as the enhancement of regulatoryT cell responses, this therapeutic strategy can be more effective anddurable than non-targeted anti-inflammatory cytokines. Compared tonon-targeted methods, targeted method described herein requires muchless amount of IL-10 to show the same or similar effects. With thedose-sparing-effect along with the delivery of IL-10 to subsets ofpatient's APCs, this strategy is expected to significantly reduce sideeffects of anti-inflammatory cytokine treatment, but with bettereffects.

To study the effects of APC-targeted IL-10, recombinant fusion proteinof antibody and human IL-10 were made. Monoclonal antibodies (anti-CD40clone 12E12, anti-CD40 clone 24A3, anti-DCIR clone 9E8, anti-DC-ASGPRclone 49C11, and control IgG4) and human IL-10 fusion proteins weremade.

It was found that different antibodies fused to human IL-10 can targetsubsets of human DCs in distinct patterns. The ability of antibody-IL-10fusion proteins to bind to human DCs was measured. Both myeloid DC(mDCs) and plasmacytoid DCs (pDCs) were purified from human blood. DCswere incubated for 20 min in ice in the presence of differentconcentrations of recombinant fusion proteins of antibody and IL-10(FIG. 1). After vigorous washing, DCs were further stained withanti-human IL-10 to detect surface bound antibody-IL-10 fusion proteinsusing flow cytometry. As shown in FIG. 1, all the recombinant fusionproteins of antibody-IL-10 (except for the control IgG4-IL-10) couldbind to mDCs. However, binding patterns of the individual proteins tomDCs were not the same. For example, anti-DCIR (9E8)-IL-10 binds to mDCsmore efficiently than do the others. In addition, anti-CD40(12E12)-IL-10 shows better binding to mDCs than anti-CD40 (24A3)-IL-10.Although both anti-CD40 (12E12)-IL-10 and anti-CD40 (24A3)-IL-10 bind topDCs, anti-DCIR (9E8)-IL-10 shows the besting binding to pDCs.

Taken together, these data indicate that anti-inflammatory cytokines(including IL-10) fused to different antibodies can target differentsubsets of human APCs in different levels, which can result in differentoutcomes of immune responses.

It was also found that antibody-IL-10 fusion proteins can suppress DCmaturation. DCs are the major APCs that can induce and direct hostimmune responses toward either immunity or tolerance. It is also knownthat matured DCs induce immunity whereas immatured DCs induce immunetolerance. Therefore, the effectiveness of antibody-IL-10 fusionproteins on the maturation of DCs induced by Escherichia colilipopolysaccharide (LPS: toll-like receptor 4 ligand) was tested.Purified blood mDCs were cultured overnight with 0, 10, and 100 ng/mlLPS in the presence or absence of 10 μg recombinant fusion proteinsindicated or the same molar concentration of recombinant IL-10. mDCswere then stained with anti-CD83 and anti-CD86 to measure the expressionlevels of these two surface molecules (indicators for DC maturation)using flow cytometry. FIG. 2 shows that untargeted human IL-10 slightlydecreased CD83 and CD86 expression. Compared to untargeted IL-10,targeted delivery of IL-10 using recombinant fusion proteins of antibodyand IL-10 were far more efficient to suppress the LPS-induced DCmaturation. Anti-CD40 (12E12)-IL-10 was slightly more efficient thanothers to suppress the expression of CD86.

These data demonstrate that recombinant fusion proteins of antibody andIL-10 can efficiently target human DCs and thus can effectively suppressDC maturation. This indicates that targeted delivery ofanti-inflammatory cytokines to human APCs can efficiently suppressongoing inflammatory responses by the inhibition of APC, including DCs,maturation.

It was next found that targeted delivery of IL-10 to DCs usingrecombinant fusion proteins of antibody and IL-10 can efficientlysuppress T cell responses. The effectiveness of recombinant fusionproteins of antibody and IL-10 in T cell responses (FIG. 3) was furtherassessed. Purified mDCs were incubated for 2 h with differentconcentrations of either recombinant human IL-10 or fusion proteins ofantibody and IL-10. CFSE-labeled allogeneic CD4+ T cells wereco-cultured for 5 days and T cell proliferation was assessed bymeasuring CFSE dilution with flow cytometry. Compared to IL-10 alone,recombinant fusion proteins of antibody and IL-10 were far moreefficient to suppress the allogeneic CD4+ T cell proliferation. Toresult in 50% inhibition of T cell proliferation, 54 nM IL-10 wasrequired, while only less than 5.4-0.054 nM (IL-10) was required toresult in similar effect on T cell proliferation when IL-10 wasdelivered to DCs in a targeted fashion.

Taken together, the data (FIGS. 1, 2, 3) demonstrate that 1) recombinantfusion proteins of antibody and anti-inflammatory cytokines caneffectively target human APCs with different patterns, depending on APCsubsets (FIG. 1); 2) they can suppress human APCs (including DCs)activation and maturation; and 3) can effectively suppress T cellresponses.

Example 2: Treating GVHD with Anti-DC-ASGPR

Tolerance to specific antigens is the ultimate goal for the success oftransplantation. Over the past several decades, a large array ofimmunosuppressive agents has been developed and is being used forpatients. However, immunosuppression does not guarantee the preventionof alloreaction over time in patients who receive organs, tissues, andhematopoietic stem cell (HPSC) transplantation. As a consequence,patients succumb to graft-versus-host disease (GVHD) as well as seriousside effects due to life-long immunosuppression. T cell depletion alsocompromises the graft-versus-leukemia (GVL) effects in alloHPSCtransplantation. Furthermore, controlling GVHD with nonspecificimmunosuppression neither spares pre-existing memory cells nordiscriminates between alloreactive and non-alloreactive T cells. Thus,although GVHD could be controlled to some degree by immunosuppression,it is at the cost of increased incidence of graft failure, leukemiarelapse, and compromised immunity to post-transplant infections, such ascytomegalovirus (CMV). Therefore, a new therapeutic strategy that canprevent GVHD while preserving host immunity to infections will bringgreat benefit to patients.

Dendritic cells (DCs), major antigen presenting cells (APCs), can inducehost immune responses. DCs also display functional plasticity to controlimmune responses. The ability of DCs, as immune controllers, is in partby the expression of pattern-recognition receptors (PRRs), includinglectins. It was discovered that a lectin expressed on human DCs,DC-asialoglycoprotein receptor (DC-ASGPR), shows a unique ability togenerate antigen-specific IL-10-producing regulatory T cells (Tregs).This applies to both self (prostate specific antigen) and foreignantigens (influenza HA1), as demonstrated in human in vitro andnon-human primates in vivo. DC-ASGPR-induced antigen-specific Tregsefficiently suppress effector T cell proliferation and inflammatorycytokine expression. It was further discovered that signals via DC-ASGPRinduce DCs to express IL-10, and this IL-10 promotes the generation ofantigen-specific Tregs. Applicants sought out to test whether activationof DCs via DC-ASGPR can generate alloantigen-specific Tregs and thus canprevent GVHD and allograft transplantation. Data shows that targetingDC-ASGPR with anti-DC-ASGPR antibody results in decreased allogeneic Tcell responses. These T cells can also secrete high level of IL-10during their reactivation in response to alloantigens. Thus, Applicantssurmise that DC-ASGPR can be a novel therapeutic target to inhibit suchunwanted types of immune responses in patients who undergotransplantation surgery. This strategy is focusing on the induction ofalloantigen-specific Tregs and thus may not interfere with host immunityto post-transplantation infections. Therefore, it was hypothesized thattargeting DC-ASGPR with an anti-DC-ASGPR antibody not fused to anantigen prevents GVHD and allograft rejection but does not interferewith host immunity to infections.

Establishment of alloantigen-specific immune tolerance is an ultimategoal for the success of transplantation. The novel immunotherapeuticstrategy described herein may eventually permit the production ofalloantigen-specific Tregs in patients without interfering with hostimmunity to post-transplantation infections. Therefore, this study has ahigh significance in both medical and immunological implications.

The approach to controlling GVHD and transplant rejection by targetingDC-ASGPR is highly novel and innovative in the aspects of both basicimmunology and medical implications.

DC-ASGPR has a specialized function to generate antigen-specific Tregs.DC-ASGPR, a scavenger receptor (Li, et al., 2012; Valladeau, et al.,2001), is expressed on subsets of human DCs (blood myeloid DCs: mDCs andskin dermal DCs but not plasmacytoid DCs: pDCs or Langerhans cells:LCs), monocytes, macrophages, and B cells (Li, et al., 2012).Endothelial cells express ASGPR, but not DC-ASGPR. DC-ASGPR is expressedin non-human primates (NHPs) (Li, et al., 2012), but not in mice. Micehave two closely linked genes called Mgl-1 and Mgl-2 which are distantlyrelated to human DC-ASGPR, the former having a closer tissuedistribution profile to the single human gene (not shown).

A. Anti-DC-ASGPR Antibody Treatment Suppresses Allogeneic CD4+ and CD8+T Cell Proliferation

To study the immunological function of DC-ASGPR, mouse monoclonalantibodies (mAbs) specific for human DC-ASGPR were first generated (Li,et al., 2012). To abolish their non-specific bindings to FcRs,recombinant mAbs carrying mouse variable region chimeras with human κchain and human IgG4 carrying two site mutations (Reddy, et al., 2000)were made (Li, et al., 2012). Recombinant control mAb was also made inthe same way.

It is important to note that both DC-ASGPR and Dectin-1 (Ni, et al.,2010) carry an immunoreceptor tyrosine-based activation motif (ITAM) andcan induce IL-10 expression in DCs. However, DC-ASGPR is superior toDectin-1 to generate Tregs (data not shown). In addition, anti-DC-ASGPRmAb does not induce DCs to express IL-1β, IL-23 or IL-12, whileanti-Dectin-1 mAb does induce these cytokines, as previously described(Ni, et al., 2010).

Anti-DC-ASGPR mAb can suppress MHC-mismatched allogeneic T cellresponses: The effects of anti-DC-ASGPR mAb in MHC-mismatched allogeneicT cell responses was tested (FIG. 4). Different numbers of PKH25-labeledPBMCs from healthy donors were incubated overnight in the presence ofanti-DC-ASGPR or control mAb, and then CFSE-labeled PBMCs fromMHC-mismatched donors (total 6 pairs of MHC-mismatched donors) wereco-cultured for 5 days. The percents of CFSE⁻CD4⁺ and CFSE⁻CD8⁺ T cellsare presented. In the presence of control mAb, both CD4⁺ and CD8⁺ T cellproliferations were correlated with the numbers of stimulators (PBMCsfrom other donors). However, anti-DC-ASGPR mAb significantly decreasedallogeneic CD4⁺ and CD8⁺ T cell proliferation, particularly when thenumber of stimulators (X-axis) was greater than 12.5×10³/well.Interestingly, total numbers of CD4⁺ and CD8⁺ T cells counted at the endof cultures were similar in both groups (control and anti-DC-ASGPRmAb-treated groups) (not shown).

B. IL-10 Secreted from PBMC Activated with Anti-DC-ASGPR Contributes tothe Suppression of Allogeneic CD4+ and CD8+ T Cell Responses

Applicants further found that the decreased allogeneic T cellproliferation by anti-DC-ASGPR mAb was recovered (˜60-70%) byneutralizing IL-10 on day 1 (2 h before adding MHC-mismatched PBMCs tothe culture) (FIG. 5). This suggests that IL-10 secreted fromanti-DC-ASGPR-activated APCs contributes to the decreased proliferationof T cells from MHC-mismatched donors.

C. Anti-DC-ASGPR Antibody Treatment Results in Decreased IFNg-Producing,but Increased IL-10-Producing Regulatory T Cell Responses

On day 8 of the co-culture of PBMCs from MHC-mismatched healthy donors,CFSE^(low)CD4⁺ T cells were FACS-sorted, and then restimulated for 48 hwith T cell-depleted PBMCs (from stimulators). The amounts of IL-10 andIFNγ in the supernatants were measured (FIG. 6). MHC-mismatched CD4⁺ Tcells co-cultured anti-DC-ASGPR-treated PBMCs secreted decreased amountof IFNγ but increased amount of IL-10 compared to CD4⁺ T cellsco-cultured with the same PBMC treated with control mAb. This suggeststhat treatment of PBMCs with anti-DC-ASGPR mAb promote the induction ofalloantigen-specific Tregs which could play important roles in theinhibition of GVHD and allograft rejection in vivo.

D. Anti-DC-ASGPR Antibody Treatment Results in the Suppression of GVHDIn Vivo

Applicants further assessed the in vivo effects of anti-DC-ASGPR mAb.NOD/SCID/γc^(−/−) (NOG) mice (5 mice/group) were injected intravenously(i.v.) on day 0 with 50×10⁶ PBMCs from healthy donors. Animals alsoreceived 3 i.v. doses of antibodies (250 μg/dose) or PBS on days 0, 2,and 4. FIG. 7 shows that anti-DC-ASGPR treatment resulted in enhancedsurvival of animals (p<0.001) compared to control IgG or PBS treatment.

Taken together, this data demonstrates that targeting DC-ASGPR withanti-DC-ASGPR mAb promotes antigen-specific Treg responses. It iscontemplated that this could also apply to the in vivo establishment ofalloantigen-specific Tregs. This data and methodology described hereinis useful in the research and development of a novel therapeutic thatcan efficiently inhibit GVHD and allograft rejection without interferingwith host immune responses to infections.

Applicants focused on novel antibodies that bind the DC-ASGPR that caninduce DCs to secrete IL-10 and to induce IL-10-producingalloantigen-specific Tregs in the presence of alloantigens. Therefore,the strategy to inhibit GVHD and allograft rejection is based on twodistinct but compensatory mechanisms (FIG. 8). First (Direct Pathway),IL-10 secreted from DC-ASGPR-activated DCs will directly inhibitallogeneic T cell responses in the early time point, as shown in FIG. 5.Second (Indirect Pathway), DC-ASGPR-induced IL-10 can contribute to theinduction of IL-10-producing alloantigen-specific Tregs, as shown inFIG. 6. These two pathways could result in the enhanced survival ofhuman PBMC-transferred NOG mice (FIG. 7). Such alloantigen-specificTregs express IL-10 when they are activated at the place wherealloantigens are available in vivo (Sagoo, et al., 2011).

It is specifically contemplated that embodiments of the invention mayinclude one or more elements listed or exclude one or more elementslisted throughout the specification. For example, specific embodimentsmay include one specific item listed (e.g. antibody framework) asdescribed herein or embodiments of the invention may encompass multipleitems from a specific list, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30,40, or more. The invention may also exclude one or more listed elements,for example, some embodiments exclude 2, 3, 4, 5, 6, 7, 8, 9, 10, 20,30, 40, or more elements listed. Furthermore, when ranges or numericalvalues are provided, it is specifically contemplated that certain rangesor numerical values may be excluded from the invention. Last, when theinventions is described in terms of including a particular feature, itis specifically contemplated that the invention may also exclude suchfeature.

All of the methods disclosed and claimed herein can be made and executedwithout undue experimentation in light of the present disclosure. Whilethe compositions and methods of this invention have been described interms of preferred embodiments, it will be apparent to those of skill inthe art that variations may be applied to the methods and in the stepsor in the sequence of steps of the method described herein withoutdeparting from the concept, spirit and scope of the invention. Morespecifically, it will be apparent that certain agents which are bothchemically and physiologically related may be substituted for the agentsdescribed herein while the same or similar results would be achieved.All such similar substitutes and modifications apparent to those skilledin the art are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

What is claimed is:
 1. A method for preventing or treating graft versushost disease (GVHD) in a subject in need thereof comprisingadministering to the subject a pharmaceutical composition comprising atherapeutically effective amount of an anti-DC-ASGPR antibody or antigenbinding fragment thereof operatively linked to IL-10; wherein thepharmaceutical composition does not comprise an antigen associated withan autoimmune disease or inflammatory condition.
 2. The method of claim1, wherein the anti-DC-ASGPR antibody or antigen binding fragmentthereof comprises three heavy chain CDRs and three light chain CDRs fromthe variable regions of an anti-DC-ASGPR heavy chain and light chainvariable region pair selected from SEQ ID NO:3 and 8; SEQ ID NO:58 and60; SEQ ID NO:62 and 64; or SEQ ID NO:66 and
 68. 3. The method of claim1, wherein the anti-DC-ASGPR antibody or antigen binding fragmentthereof comprises a γ4 constant region comprising a substitution ofglutamic acid at residue 235 and/or a substitution of proline at residue228 in the hinge region.
 4. The method of claim 1, wherein the subjecthas been diagnosed as having GVHD.
 5. The method of claim 1, wherein thesubject is one that will receive or has received transplanted tissue. 6.The method of claim 1, wherein the method comprises administering to thesubject an anti-DC-ASGPR antibody.
 7. The method of claim 1, wherein theanti-DC-ASGPR antibody or antigen binding fragment thereof isadministered intravenously.